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Plant Cell Physiol, 2004 Dec, 45(12), 1889 - 94
Peroxisomal Localization of Sulfite Oxidase Separates it from Chloroplast-based Sulfur Assimilation; Nowak K et al.; Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein . The purpose of the present study was to determine the subcellular localization of this novel plant enzyme . Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes . To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves . Our results showed a punctate fluorescence pattern resembling that of peroxisomes . Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting . By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO . This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.

Biochem Biophys Res Commun, 2005 Feb 25, 327(4), 1155 - 62
The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth; Shim BS et al.; The two-kringle domain of tissue-type plasminogen activator (t-PA) has previously been shown to contain anti-angiogenesis activity . In this study, we explored the potential in vivo anti-tumor effects of the recombinant kringle domain (TK1-2) of human t-PA . Anti-tumor effects of purified Pichia-driven TK1-2 were examined in nude mice models by subcutaneous implantation of human lung (A-549) and colon (DLD-1, HCT-116) cancer cell lines . Mice bearing the tumors were injected with PBS or purified TK1-2 (30mg/kg) i.p . every day for 22 days . TK1-2 treatment suppressed the A-549, DLD-1, and HCT-116 tumor growth by 85.3%, 52.4%, and 62.5%, respectively . Immunohistological examination of the tumor tissues showed that TK1-2 treatment decreased the vessel density and also the expression of angiogenesis-related factors including angiogenin, VEGF, alpha-SMA, vWF, and TNF-alpha, and increased the apoptotic fraction of cells . TK1-2 neither inhibited in vitro growth of these cancer cells nor affected t-PA-mediated fibrin clot lysis . These results suggest that TK1-2 inhibits the tumor growth by suppression of angiogenesis without interfering with fibrinolysis.

J Biotechnol, 2005 Mar 2, 116(1), 35 - 50 Epub 2004 Dec 10.
Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation; Oliveira R et al.; In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity . The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level . This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate . In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P . pastoris cultivation in complex media . The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters . Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain . The latter alternative proved to be more robust to errors in the measurements of the off-gas composition . Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed . It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.

Yi Chuan Xue Bao, 2004 Nov, 31(11), 1182 - 9
Expression of human soluble gp190 in yeast Pichia pastoris; Li G et al.; The cDNA of N-terminal extracellular domain of human leukemia inhibitory factor receptor a-subunit (gp190) ,which encoded soluble gp190 (sgp190) ,was cloned into the methylotrophic yeast Pichia pastoris secreted expression vector pPIC9K . The linearized plasmid sgp190-pPIC9K was then integrated with GS115 genome by spheroplasts transformation . MD medium and PCR method were used for screening positive transformants . The transformants (His+ Mut+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418 and induced by 0.5% methanol in shaking flask to express sgp190 . The sgp190 expressed level could reach up to 26% of total proteins in culture supernatant . SDS-PAGE and Western blotting analysis showed that the molecular weight of sgp190 is about 125 kD and could be specifically recognized by polyclonal antibodies against human sgp190,which indicated good antigenicity of this secreted expressed protein . Biological activity assay showed that purified sgp190 could inhibit the effect of LIF on hCG biosynthesis of cytotrophoblast in vitro.

Microbiol Res, 2004, 159(4), 331 - 8
Yeasts as biological agents to control Botrytis cinerea; Santos A et al.; Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains . A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B . cinerea strains . Pichia membranifaciens, P . anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains . In small-scale trials, post-harvest application of P . membranifaciens CYC 1106 to apple wounds inhibited B . cinerea CYC 20010 . Purified killer toxin from P . membronifaciens CYC 1106 inhibited B . cinerea CYC 20010 . Results indicated that certain yeasts, or their toxins such us P . membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B . cinerea.

Ital J Biochem, 2004 Jul, 53(2), 67 - 72
Expression and characterization of human tumor necrosis factor-related apoptosis inducing ligand in methylotrophic yeast Pichia pastoris; Wang J et al.; Recombinant human TRAIL was successfully expressed in a secreted form in methyltrophic yeast Pichia pastoris induced by methanol . The expressive product was immunoreactive to TRAIL antibody . The addition of the expressive product into cultured human lung cancer cell A549 showed moderate cell death, typical morphological characterization of apoptotic bodies, and DNA fragmentation . This result provided a new method to produce recombinant human TRAIL as a cancer therapeutic drug.

Protein Expr Purif, 2005 Feb, 39(2), 144 - 51
High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris; Li L et al.; Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi . Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris . The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418 . Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR . When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100Da recombinant CHP (rCHP) expression product . Large scale expression revealed that rCHP was produced at 108mg/L under optimal conditions in the highest Chp-producing P . pastoris clone . The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi . Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity . Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.

Ai Zheng, 2005 Jan, 24(1), 33 - 9
{Expression of epidermal growth factor-adenovirus e4orf4 fusion protein in tumor cells and its cytotoxicity.}; Bu PL et al.; BACKGROUND & OBJECTIVE: Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation . EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells . Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells . Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells . METHODS: EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR . EGF-E4orf4 fragment was cloned into the yeast expression vector . Recombinant plasmid DNA was transformed into the yeast Pichia pastoris . Fusion proteins were purified by SP Sepharose ion exchange chromatography . Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry . RESULTS: The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4 . Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes . Apparent molecular weight of fusion protein was 20 ku . Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody . EGF-E4orf4 in high concentrations (5, and 0.5 mug/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls . Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 mug/3x10(5) cells . CONCLUSIONS: Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.

Gen Comp Endocrinol, 2005 Feb, 140(3), 222 - 32 Epub 2004 Dec 15.
Production of biologically active tethered tilapia LHbetaalpha by the methylotrophic yeast Pichia pastoris; Kasuto H et al.; In fish, luteinizing hormone (LH) stimulates processes leading to final oocyte maturation and ovulation in females, and spermiation in males . The hormone is a heterodimeric glycoprotein composed of two non-covalently associated subunits . In this study, we describe the expression of tilapia LH (tLH) as a biologically active, single-chain polypeptide using the methylotrophic yeast Pichia pastoris . The tLHbeta and alpha mature protein-coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which the tLHbeta-chain forms the N-terminal part and the alpha-chain forms the C-terminal part . A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the beta- and alpha-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the beta-subunit, to enable purification of the recombinant protein . Western blot analysis of the pituitary LH resolved by SDS-PAGE yielded a band of 35kDa, while the recombinant tLHbetaalpha had a molecular mass of 45kDa, and was found to possess only N-linked carbohydrates . Recombinant tLHbetaalpha stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.

Appl Microbiol Biotechnol . 2005 Jan 6; {Epub ahead of print}
Xylitol production by a Pichia stipitis D: -xylulokinase mutant; Jin YS et al.; Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Delta1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions . Under both culture conditions, FPL-YS30 (xyl3-Delta1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose) . However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions . This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration . As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l(-1) h(-1)) under aerobic conditions.

Microbiology, 2005 Jan, 151(Pt 1), 145 - 55
Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum; Monod M et al.; The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach . T . rubrum genomic and cDNA libraries were screened with Aspergillus spp . and Saccharomyces cerevisiae aminopeptidase genes as the probes . Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris . RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein . The hydrolytic activity of ruLap1, ruLap2 and A . fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations . ruDppIV and ruDppV showed similar activities to A . fumigatus orthologues . In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T . rubrum in a medium containing keratin as the sole nitrogen source . Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Appl Microbiol Biotechnol . 2005 Jan 4; {Epub ahead of print}
Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain; Pitkanen JP et al.; The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development . This study focused on the recovery and characterization of xylose chemostat isolates of a S . cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase . The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source . Under aerobic conditions, on minimal medium with 30 g l(-1) xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h(-1) vs 0.05 h(-1)) . In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism . The xylose uptake rate was increased almost 2-fold . Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly . Under anaerobic conditions, on minimal medium with 45 g l(-1) xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g(-1) CDW h(-1) to 0.017 g g(-1) CDW h(-1) and the yield from 0.09 g g(-1) xylose to 0.14 g g(-1) xylose, respectively.

Arch Biochem Biophys, 2005 Feb 1, 434(1), 201 - 11
Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands; Cristea M et al.; Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis . We expressed the enzyme in Pichia pastoris, which secreted approximately 30mg Mn-lipoxygenase/L culture medium in fermentor . The recombinant lipoxygenase was N- and O-glycosylated (80-100kDa), contained approximately 1mol Mn/mol protein, and had similar kinetic properties (K(m) approximately 7.1muM alpha-linolenic acid and V(max) 18nmol/min/mug) as the native Mn-lipoxygenase . Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein ( approximately 67kDa) with retention of lipoxygenase activity (K(m) approximately 6.4muM alpha-linolenic acid and V(max) approximately 12nmol/min/mug) . Putative manganese ligands were investigated by site-directed mutagenesis . The iron ligands of soybean lipoxygenase-1 are two His residues in the sequence HWLNTH, one His residue and a distant Asn residue in the sequence HAAVNFGQ, and the C-terminal Ile residue . The homologous sequences of Mn-lipoxygenase are H(274)VLFH(278) and H(462)HVMN(466)QGS, respectively, and the C-terminal amino acid is Val-602 . The His274Gln, His278Glu, His462Glu, and the Val-602 deletion mutants of Mn-lipoxygenase were inactive, and had lost >95% of the manganese content . His-463, Asn-466, and Gln-467 did not appear to be critical for Mn-lipoxygenase activity, as His463Gln, Asn466Gln, Asn466Leu, and Gln467Asn mutants metabolized alpha-linolenic acid to 11- and 13-hydroperoxylinolenic acids . We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.

J Biol Chem . 2004 Dec 28; {Epub ahead of print}
Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc; Watanabe S et al.; Pichia stipitis NAD+-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase (MDR), is one of the key enzymes in ethanol fermentation from xylose . For the construction of an efficient biomass-ethanol conversion system, we focused on the two following areas of XDH: (1) change of coenzyme specificity from NAD+ to NADP+, (2) thermostabilization by introducing an additional zinc atom . Site-directed mutagenesis was used to examine the roles of Asp207, Ile208, Phe209 and Asn211 in the discrimination between NAD+ and NADP+ . Single mutants (D207A, I208R, F209S and N211R) improved 5~48-fold in catalytic efficiency (kcat/Km) with NADP+, compared with the wild type, but retained substantial activity with NAD+ . The double mutants (D207A/I208R and D207A/F209S) improved by three orders of magnitude in kcat/Km with NADP+, but they still preferred NAD+ to NADP+ . The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed over 4,500-fold higher values in kcat/Km with NADP+ than the wild-type enzyme, reaching values comparable to kcat/Km with NAD+ of the wild-type enzyme . Since most NADP+-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom . The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability . The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability, and further increased the catalytic activity with NADP+.

J Virol Methods, 2005 Feb, 123(2), 221 - 5
Secreted expression of the VP2 protein of very virulent infectious bursal disease virus in the methylotrophic yeast Pichia pastoris; Wu PC et al.; The VP2-encoding gene of very virulent infectious bursal disease virus (vvIBDV) was amplified using reverse transcription (RT)-polymerase chain reaction (PCR) and inserted into pPICZalphaA vector . Recombinant plasmid DNA was integrated into the chromosome of the transformed Pichia pastoris by electroporation and expressed protein identified by SDS-PAGE and Western blotting . High-level secreted expression was performed by determining the Mut(+) phenotype and secreting multi-copy integrants in the recombinant yeast . A recombinant protein of approximately 67kDa was secreted into the supernatant from the yeast when induced with methanol . The expressed supernatant was bound with chicken anti-IBDV polyclonal antibodies . Western blotting with antibodies against vvIBDV indicated that the recombinant VP2 protein retained its antigenicity . High-level production (10mg/100ml) of the recombinant VP2 protein indicated that P . pastoris was an efficent expression system for vvIBDV VP2 protein.

Mol Hum Reprod . 2004 Dec 22; {Epub ahead of print}
Psoriasin, a calcium-binding protein with chemotactic properties is present in the third trimester amniotic fluid; Porre S et al.; Psoriasin is a small calcium-binding protein first found in psoriatic lesions and also up-regulated in other inflammatory skin diseases and cancer tissues . Psoriasin is also present in the fetal epithelial cells . Its biological function is unclear, but there is both in vitro and in vivo evidence for its chemokine-like activity . The aim of the present study was to find whether psoriasin could be found in the amniotic fluid and thus could have long-range immunobiological effects . Two recombinant psoriasins were prepared, one in Escherichia coli, the other one in Pichia pastoris . The former was used to produce a rabbit antiserum against psoriasin . Fractionation of full-term amniotic fluids with polyacrylamide gel electrophoresis (PAGE) and gel filtration associated with immunodetection with the antiserum were used to identify a protein compatible with the size of psoriasin . The identity of psoriasin was further verified by mass spectrometric analysis . Expression of psoriasin in cells of the amniotic membranes was detected with nested RT-PCR . Because of its chemokine-like activity, psoriasin present in the amniotic fluid might have consequential immunobiological effects during the fetal development.

Mol Biol (Mosk), 2004 Nov-Dec, 38(6), 1067 - 75
{Production of DNA-hydrolyzing antibody BV04-01 Fab fragment in metylotrophic yeast Pichia pastoris}; Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation; BACKGROUND: Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty . The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P . pastoris from the beginning . Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P . pastoris cDNA . RESULTS: We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P . pastoris by probing S . cerevisiae DNA microarrays . The number of positive signals was about 66 % as compared to homologous S . cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P . pastoris and S . cerevisiae samples . The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail . Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified . CONCLUSIONS: We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P . pastoris . The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase.

Biochem Biophys Res Commun, 2005 Jan 28, 326(4), 817 - 24
Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations; Liu SH et al.; Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris . The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA . The P . pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1 . Moreover, the P . pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1 . In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P . pastoris transformant . Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1 . In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R . oryzae GA in P . pastoris.

Biotechnol Lett, 2004 Oct, 26(20), 1569 - 73
Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica; Wang H et al.; The cDNA encoding for lignin peroxidase of Phanerochaete chrysosporium was expressed in the Pichia methanolica under the control of the alcohol oxidase (AUG1) promoter which was followed by either the lignin peroxidase leader peptide of Phanerochaete chrysosporium or the Saccharomyces cerevisiae alpha-factor signal peptide . Both peptides efficiently directed the secretion of lignin peroxidase from the recombinant yeast cell . The extracellular lignin peroxidase activity in two recombinants was 932 U l(-1) and 1933 U l(-1) . The purity of the recombinant product was confirmed by SDS-PAGE.

Biotechnol Lett, 2004 Oct, 26(19), 1475 - 9
High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization; Seo KH et al.; The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris . The phospholipase C (PLC) when expressed in P . pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium . Recombinant P . pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P . pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein) . The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P . pastoris GS115, 1176 U mg(-1) protein by P . pastoris KM71H and 1522 U mg(-1) protein by P . pastoris X-33 . The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g . stable at 75 degrees C) than the wild-type PLC from B . cereus . Some organic solvents, surfactants and metal ions, e.g . methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.

Biotechnol Lett, 2004 Sep, 26(18), 1447 - 52
Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris; Ramon R et al.; The Pichia pastoris expression system is widely used for the production of recombinant proteins . A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P . pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described . An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed . Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively . The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.

Plant Mol Biol, 2004 Sep, 56(2), 285 - 98
Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1; Tang CM et al.; We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains . Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense . In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing . To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris . Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using alpha-mannosidase . Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by alpha-mannosidase . BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases . Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3 . To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3 . Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity . H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis . E234A showed 36% retention of activity and substitution Y269D, 50% . The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively . Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.

Plant Mol Biol, 2004 Jul, 55(5), 631 - 44
A genetic and structural analysis of the N-glycosylation capabilities; Leonard R et al.; The recent draft sequencing of the rice (Oryza sativa) genome has enabled a genetic analysis of the glycosylation capabilities of an agroeconomically important group of plants, the monocotyledons . In this study, we have not only identified genes putatively encoding enzymes involved in N-glycosylation, but have examined by MALDI-TOF MS the structures of the N-glycans of rice and other monocotyledons (maize, wheat and dates; Zea mays, Triticum aestivum and Phoenix dactylifera); these data show that within the plant kingdom the types of N-glycans found are very similar between monocotyledons, dicotyledons and gymnosperms . Subsequently, we constructed expression vectors for the key enzymes forming plant-typical structures in rice, N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), core alpha1,3-fucosyltransferase (FucTA; EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) and successfully expressed them in Pichia pastoris . Rice GlcNAc-TI, FucTA and xylosyltransferase are therefore the first monocotyledon glycosyltransferases involved in N-glycan biosynthesis to be characterised in a recombinant form.

Plant Mol Biol, 2004 May, 55(3), 343 - 67
Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1; Xu Z et al.; In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels . Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant . Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes . Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members . Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations . The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage . Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented . To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system . To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P . pastoris and purified to homogeneity . When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A . thaliana in degradation of mannans, galactomannans, or glucogalactomannans . Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

Glycobiology . 2004 Dec 15; {Epub ahead of print}
Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates; Paschinger K et al.; Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates . In order to prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologues of vertebrate alpha1,6-fucosyltransferases (EC 2.4.1.68) were engineered for expression in the yeast Pichia pas oris . Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods.Unsubstituted non-reducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha1,6- fucosyltransferases, as well as for native Caenorhabditis and Schis osoma mansoni core alpha1,6-fucosyltransferases . On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to utilise core alpha1,6-fucosylated glycans as substrates . Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3- fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase . Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 259 - 63
Human, monoclonal and recombinant candidacidal, pneumocysticidal and mycobactericidal antibodies; Polonelli L; Antiidiotypic antibodies (antiIds) representing the internal image of some antigenic deteminants have been proposed as surrogate vaccines or, conjugated with toxins, in the immunotherapy of cancer . Experimental studies on antiidiotypic vaccination against fungal infections have been lacking . A conceptually new model of idiotypic vaccination against fungal infection has been recently developed . The vaccine used is monoclonal antibody that in vitro neutralizes the activity of killer toxin from the yeast, Pichia anomala (KT) . This is effective against a wide range of fungal pathogens including Candida albicans and Pneumocystis carinii, and is also active against Mycobacterium tuberculosis . In an experimental mouse model, this vaccination was found to confer significant protection against systemic and mucosal infection with C . albicans . Human recombinant killer toxin antibodies and its synthetic derivatives hold promise of a potentially powerful tool against fungal and mycobacterial infections.

Protein Expr Purif, 2005 Jan, 39(1), 35 - 42
High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris; Masuda T et al.; Hen egg lysozyme (HEL) is one of the sweet-tasting proteins . To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method . The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha . This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast . Expression of HEL was carried out in fermenter cultures . Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography . Approximately 400mgL(-1) of recombinant HEL was obtained . The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans . The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus . These results demonstrate that the P . pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.

Appl Microbiol Biotechnol . 2004 Dec 9; {Epub ahead of print}
Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris; Chang SW et al.; Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications . Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization . In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C . rugosa LIP1 in the Pichia expression system . Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P . pastoris . Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6 . The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml . The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

Eukaryot Cell, 2004 Dec, 3(6), 1567 - 73
Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family; Shalguev VI et al.; The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51(Pa)) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes . We cloned the RAD51 gene from the cDNA library of the thermotolerant P . angusta strain BKM Y1397 . Induction of this gene in a rad51-deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation . Purified Rad51(Pa) protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51(Pa) per 3 nucleotides) and DNA specificity for ssDNA-dependent ATP hydrolysis {poly(dC) > poly(dT) > phiX174 ssDNA > poly(dA) > double-stranded M13 DNA} . An inefficient ATPase and very low cooperativity for ATP interaction position Rad51(Pa) closer to Rad51 than to RecA . Judging by thermoinactivation, Rad51(Pa) alone was 20-fold more thermostable at 37 degrees C than its S . cerevisiae homolog (Rad51(Sc)) . Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees C . A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51(Pa) occurred only at temperatures above 42 degrees C . These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.

Biol Res, 2004, 37(4 Suppl A), 747 - 57
Genetic polymorphism of clinical and environmental strains of Pichia anomala; Reyes E et al.; In this work 20 clinical and 3 environmental yeast isolates were characterized by classical morphological and physiological methods, as well as molecular methods based on PCR of the ITS1-5.8S rDNA-ITS2 region . The characteristic morphology and biochemical profiles observed in these samples correspond to those described for the Pichia genera, more specifically to P . anomala . The profiles of susceptibility to five antifungal drugs were determined by two broth dilution methods . The results obtained by both methods were comparable and showed that clinical isolates presented more resistance to azoles, amphotericin B, and 5-fluorocytosine, than environmental ones did . By amplification and sequencing of internal transcribed spacers (ITS1 and ITS2) and the ribosomal 5.8S DNA, the yeast samples were divided into four groups, where the strains within each group had the same sequence . Of the analyzed yeast isolates, 78% were identified as Pichia anomnala . Using RAPD analysis with seven different Operon primers, polymorphism was observed within the four groups . Our study highlights the growing importance of P . anornala in fungemic episodes in premature neonates . Furthermore, the methodologies used provide a powerful tool to identify and determine differences in similar strains of this yeast.

J Virol Methods, 2005 Jan, 123(1), 35 - 40
Expression and characterization of Gag protein of HIV-1(CN) in Pichia pastoris; Jiang WZ et al.; To express the core protein of HIV-1 of Chinese prevalent strain (HIV-1(CN)) in Pichia pastoris, the full-length gag gene was inserted into the secretory expression vector pHILS1 . Linearized recombinant plasmid pHILGAG by SalI was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR . To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning . mRNA (1.3kb) was amplified by RT-PCR . SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24 . Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant . The recombinant yeast had good genetic stability . The optimal expression conditions of the engineering yeast were as follows: BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course . Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography . The purity of the interest protein was up to 85% . After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.

Biotechnol Bioeng, 2005 Jan 5, 89(1), 102 - 12
Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau; Sinha J et al.; It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped . Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h . Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol . It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein . (c) 2004 Wiley Periodicals, Inc.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1766 - 75
Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris; Trentmann O et al.; A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter . In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared . After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate . Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable . To postpone the onset of limitation, the inlet air was enriched by pure oxygen . Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period . Without additional oxygen supply, the process was robust . Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems . Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.

Traffic, 2005 Jan, 6(1), 66 - 74
Atg8 is Essential for Macropexophagy in Hansenula polymorpha; Monastyrska I et al.; We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis . Sequencing revealed that the mutant was affected in the HpATG8 gene . HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy . Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole . After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome . In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained . We hypothesize that H . polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.

J Mol Recognit . 2004 Nov 26; {Epub ahead of print}
Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production; Daly R et al.; The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times . P . pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities . Equally important, P . pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality . Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed . A further benefit of the P . pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems . The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein . Included in these considerations are the influences of P . pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P . pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers . The impact of the alcohol oxidase (AOX) pathways in terms of the mut(+) and mut(s) phenotypes, intracellular expression and folding pathways is examined . The roles of pre-pro signal sequences such as the alpha mating factor (alpha-MF) and the Glu-Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered . Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described . Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered . Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed . Copyright (c) 2004 John Wiley & Sons, Ltd.

Yeast, 2004 Dec, 21(16), 1325 - 34
Bax-induced cell death in Candida albicans; De Smet K et al.; Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells . It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death . In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans . Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans . Expression of this BAX gene resulted in growth inhibition and cell death . By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax . Using this fusion protein we showed that, upon expression in C . albicans, Bax co-localizes with the mitochondria . Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region.

Yeast, 2004 Dec, 21(16), 1343 - 58
Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe; Kumar R et al.; Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus . Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded . Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast . A chimeric gene comprising the signal sequence of the Plus pheromone of Sz . pombe fused in-frame with the mature streptokinase from Streptococcus sp . was constructed and inserted into the expression vector containing the thiamine-regulated promoter . We obtained a high level of expression of streptokinase comparable to that in E . coli and P . pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction . The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional . Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz . pombe defective for a potent extracellular protease activity . We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz . pombe.

Int Arch Allergy Immunol, 2004 Dec, 135(4), 284 - 92 Epub 2004 Nov 24.
Che a 1: recombinant expression, purification and correspondence to the natural form; Barderas R et al.; BACKGROUND: Pollinosis to chenopods is one of the main causes of allergy in desertic regions and it is increasing in the South of Europe and Western USA . Che a 1 is a major allergen for chenopod-allergic subjects and belongs to the Ole-e-1-like family of proteins . METHODS: Pichia pastoris yeast has been used as expression system to produce the recombinant form of Che a 1 (rChe a 1) . The allergen was isolated using a gel permeation column and reverse-phase/high-performance liquid chromatography . Molecular characterization was performed using Edman degradation, mass spectrometry and concanavalin A staining . Sera from patients allergic to chenopod pollen, as well as polyclonal and monoclonal antibodies raised against Ole e 1, were used in immunoblotting, ELISA and inhibition assays for immunological characterization of rChe a 1 . RESULTS: The allergen was purified to homogeneity with a final yield of 15 mg/l of cell culture and showed a glycosylated character . N-terminal amino acid sequence of rChe a 1 and molecular mass were according to those of the protein isolated from chenopod pollen . The recombinant allergen maintained the IgG and IgE epitopes of the natural allergen deduced from the immunological assays . CONCLUSIONS: Structural and in vitro immunological properties of rChe a 1 produced in P . pastoris were equivalent to those of the natural form of the allergen and, thus, it could be used in testing patients allergic to chenopods . 2004 S . Karger AG, Basel.

Mol Biol Cell . 2004 Nov 24; {Epub ahead of print}
A Sorting Nexin PpAtg24 Regulates Vacuolar Membrane Dynamics during Pexophagy via Binding to Phosphatidylinositol-3-Phosphate; Ano Y et al.; Monitoring Editor: Howard Riezman Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells . In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy . Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex . Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensible for pexophagy, functions in membrane fusion at the vacuolar surface . CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy . Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage . These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidyl inositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Protein Expr Purif, 2004 Dec, 38(2), 217 - 27
Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris; O'Leary JM et al.; The use of the methylotrophic yeast Pichia pastoris for large-scale recombinant production of proteins for therapeutic uses and/or biophysical characterisation has been gaining popularity . Here we describe the use of this organism for the production of a von Willebrand factor C domain from procollagen IIA for solution NMR studies . In this research, we specifically identified sites of O-linked glycosylation on the expressed protein, although the native protein is not glycosylated . We demonstrated that it was possible to remove the oligosaccharides by enzymatic digestion, however this approach proved to be prohibitively expensive for the scale of production required for high-resolution structural studies by NMR spectroscopy . After removal of the O-linked glycosylation sites by site-directed mutagenesis, we confirmed that the protein was no longer covalently glycosylated . However, analysis by 1H- and 13C-edited spectroscopy identified the presence of non-covalently associated glycans which were removed by lectin affinity chromatography . We have synthesised methods for the identification and removal of both covalently and non-covalently bound oligosaccharides from heterologous protein expressed in P . pastoris.

Protein Expr Purif, 2004 Dec, 38(2), 196 - 204
Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization; Shrestha B et al.; The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues . The expressed enzyme was recovered from the culture supernatant and further characterized . A single-step purification based on specific binding of the histidine residues was achieved . The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry . The activity of the recombinant chitin deacetylase on chitinous substrates was investigated . With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively . The specific activity of the pure protein is 72 U/mg . One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed . The enzyme activity is enhanced in the presence of Co2+ ions . A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.

Appl Microbiol Biotechnol, 2005 Feb, 66(5), 527 - 35 Epub 2004 Nov 12.
Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II with high hydrolytic activity on sophorose; Bhatia Y et al.; beta-Glucosidase II (Bgl II), encoded by the betaglu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109 . The enzyme had a molecular mass of 176 kDa and was a dimer with an apparent subunit mass of 83 kDa . It exhibited broad substrate specificity and hydrolyzed beta-linked gluco-disaccharides and oligosaccharides, salicin, and cyanogenic glucoside amygladin . The unusually high hydrolytic activity of 7,680 units min(-1) g(-1) protein was obtained on sophorose . Competition experiments performed using differently linked beta-disaccharides indicated these to be hydrolyzed at the same active site . Transglycosylation activity leading to the biosynthesis of several disaccharides and oligosaccharides was observed . The enzyme was placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data . The involvement of His as a catalytically important residue was confirmed by diethylpyrocarbonate modification . Pre-incubation of the purified enzyme with 5 mM p-nitrophenyl-beta-D: -glucoside offered 2.5-fold higher residual activity compared with unbound enzyme, indicating protection at the active site . The feasibility of this enzyme as a biocatalyst of choice for the synthesis of glyco-conjugates is discussed.

Bioconjug Chem, 2004 Nov-Dec, 15(6), 1289 - 96
Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast; Nakamura S et al.; The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33 . As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant . The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type . In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one . The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system . A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL . More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC . Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.

Yeast, 2004 Nov, 21(15), 1307 - 16
Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis; Voronovsky AY et al.; Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced . The derived amino acid sequences of C . famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species . The highest identity was observed to homologues of D . hansenii CBS767, as C . famata is the anamorph of this hemiascomycetous yeast . The D . hansenii CBS767 RIB3 gene encoding specific deaminase was cloned . This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii . Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C . famata and D . hansenii RIB genes . The sequences of C . famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.

Eur Cytokine Netw, 2004 Jul-Sep, 15(3), 240 - 6
Expression of an interleukin-6 - interleukin-2 fusion protein (pIL-6-IL-2) in P . pastoris; Lin Y et al.; Interleukin-2 and interleukin-6 can stimulate the growth and proliferation of T lymphocytes and the differentiation of activated B lymphocytes respectively, and in turn enhance cellular and humoral immune responses . In this work, an expression clone using Pichia pastoris, a methylotrophic yeast strain, has been developed in order to produce large amounts of the functional recombinant fusion protein pIL-6-IL-2, which contains the mature porcine interleukin-6 peptide and the mature porcine interleukin-2 peptide . Two components of the fusion protein were connected by means of a flexible linker (Gly-Gly-Gly-Gly-Ser-Glu-Phe-Gly-Ser-Gly-Gly) . In response to 1% methanol induction, the recombinant strain GS115\9K-IL6-IL2 secreted an exogenous protein, with a molecular weight of approximately 40 kD, into the culture medium . This was confirmed to be pIL-6-IL-2 by means of SDS-PAGE and Western Blot analysis . The protein was visible on the 2nd day following methanol induction, and peaked on the 4th day . By this time, the level had reached 50 mg\L as determined using the method of Bradford . After treatment with PNGase F and analysis of the concentration of sugar, the fusion protein pIL-6-IL-2 was further confirmed to be mainly a glycoprotein with an approximately 2 kDa sugar decoration . In addition, the IL-6 and IL-2 biological activities of the fusion protein, determined by cell proliferation assays using the IL6-dependent cell line B9 and the IL2-dependent cell line CTLL-2, reached 1 x 10(5) U\mg and 8 x 10(5) U\mg, respectively . This report is the first description of fused porcine cytokines expressed in P . pastoris, which might be an interesting adjuvant product for veterinary vaccines.

Arch Biochem Biophys, 2004 Dec 15, 432(2), 205 - 11
The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans; Gonzalez LJ et al.; Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba) . It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region . The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS . The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant . O-Glycopeptides are distributed mostly towards the protein N-terminus . While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 660 - 4
C75 activates malonyl-CoA sensitive and insensitive components of the CPT system; Nicot C et al.; Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation . This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme . There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition . Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels . Paradoxically it also activates long-chain fatty acid oxidation . To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris . We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.

J Allergy Clin Immunol, 2004 Nov, 114(5), 1124 - 30
Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens; van Oort E et al.; BACKGROUND: Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult . Purified natural group 1 allergens could be an alternative for application in immunotherapy . OBJECTIVE: Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules . METHODS: Dac g 1 was cloned and expressed in the yeast Pichia pastoris . Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract . Dac g 1 variants were analyzed by N-terminal sequencing . Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests . RESULTS: Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens . Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr) . Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water . The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids . The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule . IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1 . CONCLUSIONS: Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography . Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.

World J Gastroenterol, 2004 Dec 15, 10(24), 3602 - 7
High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris; Liu RS et al.; AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein . This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS . METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN . The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His-Mut+) by electroporation . His(+)Mut(+) recombinant strains were identified by PCR and cultivated on MM/MD plates . The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied . The expression level and activation were detected by SDS-PAGE and Western-blot respectively . RESULTS: All of the recombinants were His(+)Mut(+) after transformation of P.pastoris with linearized plasmids . The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains . The final optimal concentration of methanol was 20 mL/L, the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains . The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved, and a maximum cell A at 600 nm of 62 was achieved in shake flask culture . The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum . The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in E.coli when the same rSCoVN protein quantity was used . CONCLUSION: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GS115 . The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum . This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.

Biochem Biophys Res Commun, 2004 Dec 3, 325(1), 68 - 74
In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein; Acosta-Rivero N et al.; Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs) . However, difficulties in obtaining purified mature HCcAg have limited these studies . In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions . Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids . The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm . Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process . However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.

Bioorg Med Chem, 2004 Dec 1, 12(23), 6063 - 75
The synthesis of phosphorylated disaccharide components of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448; Fairweather JK et al.; Methods for the stereoselective synthesis of alpha-(1-->2)- and alpha-(1-->3)-linked 6(II)-O-phosphomannobiosides were developed . Two strategies were successfully employed: a D-mannosyl acceptor was coupled with a phosphorylated D-mannosyl trichloroacetimidate donor, or alternatively with a differentially 6-O-protected D-mannosyl trichloroacetimidate donor which, after glycosylation, was selectively deprotected and phosphorylated . Two target phosphomannobiosides intended for use in SAR studies of the antiangiogenic drug candidate PI-88, 2-O-(6-O-phospho-alpha-D-mannopyranosyl)-D-mannopyranose and methyl 3-O-(6-O-phospho-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, were synthesized . The former is a minor component of the side-chain repeating unit of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448, whilst the latter represents a nonreducing end fragment of the phosphomannan.

Biochem Biophys Res Commun, 2004 Nov 26, 324(4), 1179 - 85
Expression and characterization of the first kunitz domain of human tissue factor pathway inhibitor-2; Kong D et al.; Human tissue factor pathway inhibitor-2 (hTFPI-2) has three kunitz domains whose structure and function are unclear . We expressed the first kunitz domain of hTFPI-2 (hTFPI-2/KD1) as functional form using Pichia pastoris and investigated its characterization . In the experiment, hTFPI-2/KD1 can inhibit the plasmin and trypsin activity and the Ki of hTFPI-2/KD1 towards plasmin (30nM) and trypsin (50nM) was determined as 10 and 7nM by chromogenic assay, respectively . hTFPI-2/KD1 can also inhibit MMP-2 and MMP-9 in zymography assay . Furthermore, the inhibition of hTFPI-2/KD1 to the Matrigel invasion by HT-1080 is also described . This study provides a method to produce hTFPI-2/KD1 efficiently and some insights into the structure and function of hTFPI-2/KD1.

Biosci Biotechnol Biochem, 2004 Oct, 68(10), 2111 - 9
Cloning and expression of a novel Trichoderma viride laminarinase AI gene (lamAI); Nobe R et al.; The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique . The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide . The protein showed 91% sequence similarity to the putative Trichoderma virens beta-1,3-glucanase BGN1, but no significant similarity to fungal beta-1,6-glucanases or beta-1,3-glucanases from other organisms . On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose . The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI . Based on these data, the probable existence of endo-beta-1,3:1,6-glucan hydrolases as a subclass of endo-beta-1,3-glucanases in some mycoparasitic fungi is suggested.

Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2040 - 3 Epub 2004 Oct 20.
Expression, crystallization and preliminary structural analysis of the ectoplasmic region of apical membrane antigen 1 from Plasmodium vivax, a malaria-vaccine candidate; Vulliez-Le Normand B et al.; Apical membrane antigen 1 (AMA1), a type 1 transmembrane protein present in the microneme organelles of Plasmodium, is a leading malaria-vaccine candidate . The ectoplasmic region of AMA1 from P . vivax has been expressed in Pichia pastoris and crystallized in two different forms: an orthorhombic form (space group P2(1)2(1)2(1), unit-cell parameters a = 54.1, b = 76.1, c = 103.9 A) and a monoclinic form (space group C2, unit-cell parameters a = 150.0, b = 53.8, c = 60.3 A, beta = 113.2 degrees ) . Native data have been collected to 2.0 A resolution for the orthorhombic form and 1.8 A for the monoclinic form . A platinum derivative was prepared for the orthorhombic and monoclinic crystals using K(2)PtCl(4) and data were collected at several wavelengths to obtain phases by the MAD technique . A partial model has been built from the electron-density maps of both forms and refinement is in progress.

Yi Chuan Xue Bao, 2004 Jun, 31(6), 552 - 7
Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter; Zhang AL et al.; The GAP gene promoter was amplified from P . pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K . The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K . pGAP9K-AS was then transformed into P . pastoris GS115 . The multi-copy integration transformant P . pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P . pastoris . The expression of angiostatin reached its peak after 4 d of culture in P . pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P . pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P . pastoris GS115 (pGAP9K-AS) . The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system . The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively . The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P . pastoris.

FEMS Yeast Res, 2004 Nov, 5(2), 179 - 89
Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena; Weis R et al.; Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task . We simplified the protocol for protein expression by P . pastoris and scaled it down to 0.5-ml cultures . Optimising standard growth conditions and procedures, programmed cell death and necrosis of P . pastoris in microscale cultures were diminished . Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants . Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

J Virol Methods, 2004 Dec 1, 122(1), 105 - 11
The expression of SARS-CoV M gene in P . Pastoris and the diagnostic utility of the expression product; Han X et al.; High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties . To express the membrane (M) protein of SARS-CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZalphaA . SDS-PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and that the expression product was similar antigenically to the natural protein . Purified recombinant M protein was used subsequently as an ELISA antigen for detection of eight serum samples screened previously by whole virus ELISA and immunofluorescence assay, and consistent results were obtained . These findings suggest that the recombinant M protein may be useful as a diagnostic reagent.

Biotechnol Lett, 2004 Aug, 26(16), 1269 - 72
Improving the expression of mini-proinsulin in Pichia pastoris; Pais-Chanfrau JM et al.; Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth . Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.

Appl Microbiol Biotechnol . 2004 Oct 5; {Epub ahead of print}
Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis; Klinner U et al.; The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system . There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism . Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited . The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension . The responses differed depending on the velocity of the decrease in oxygen tension . At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced . At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation . The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.

Clin Exp Allergy, 2004 Oct, 34(10), 1576 - 82
Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy; Saarelainen S et al.; BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts . OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy . METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting . RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract . Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001) . The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001) . A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported . About one-third of the patients reacted to Can f 2 . In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component . Aminoterminal sequencing showed this to be a previously unidentified allergenic protein . CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals . However, the two allergens are insufficient as reagents for diagnosing dog allergy.

Eur J Biochem, 2004 Oct, 271(20), 4132 - 40
Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin; Hilario E et al.; Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy . In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris . Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain . The functional and structural properties of the mutant Tms were compared to those of the wild type protein . None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay . The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+ . However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity . Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms . However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type . These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Mol Biochem Parasitol, 2004 Aug, 136(2), 257 - 64
A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis; Gounaris K et al.; We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5'-diphosphates and 5'-monophosphates, but not 5'-triphosphates . The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor . Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg2+-dependent diphosphohydrolase/apyrase and a 5'-nucleotidase . The protein sequence is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase . The enzyme was essentially monospecific for AMP among the nucleoside 5'-monophosphates and catalysed the hydrolysis of nucleoside 5'-diphosphates in the order of UDP >> ADP . The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5'-nucleotidase activity was enhanced by these additions . Kinetic analyses indicated that the enzyme exhibits allosteric behaviour . Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site . The data are discussed in terms of regulation of host purinergic signalling during infection.

J Cardiovasc Pharmacol, 2004 Sep, 44(3), 287 - 93
Biotinyl endothelin-1 binding to endothelin receptor and its applications; Saravanan K et al.; The endothelin (ET) system consists of two membrane receptor types A and B and three 21-mer isopeptides endothelin-1, endothelin-2, and endothelin-3 as ligands . This system is involved in many physiological processes such as vasomodulation, neurotransmission, embryonic development, renal function, and regulation of cell proliferation . In many pathophysiological conditions involving endothelin system, the endothelin antagonism could be a possible clinical treatment . Designing of an antagonist involves the characterization of the binding of the test compounds to the endothelin receptors . This is being carried out using radioactive ligand . A simpler and quicker method will be of great advantage . This study reports a non-radioactive method for establishing the IC50 concentrations of the ligand . This method uses biotinylated-endothelin-1 and streptavidin conjugated with horseradish peroxidase . Hydroxyl apatite gel is used for separating the bound and unbound biotin-tagged endothelin-1 . This method is applicable to detergent solubilized receptors and purified recombinant receptors . The endothelin receptor type A expressed in Pichia pastoris system has been used in this study . We show that this method is applicable in Western blot analysis of endothelin-1 and its receptor complex . This can be used to localize the receptor molecules as well.

Insect Biochem Mol Biol, 2004 Oct, 34(10), 1037 - 50
Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase; Fitches E et al.; Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover . A cDNA encoding chitinase was cloned from larvae of tomato moth (Lacanobia oleracea), a Lepidopteran pest of crops . The predicted protein contains 553 amino acid residues, with a signal peptide of 20 a.a . Sequence comparison showed 75-80% identity with other Lepidopteran chitinases . L . oleracea chitinase was produced as a functional recombinant enzyme in the yeast Pichia pastoris . A fusion protein containing chitinase joined to the N-terminus of snowdrop lectin (GNA) was also produced, to determine whether GNA could deliver chitinase to the haemolymph of Lepidopteran larvae after oral ingestion . The purified recombinant proteins exhibited similar levels of chitinase activity in vitro . Both proteins were highly toxic to L . oleracea larvae on injection, causing 100% mortality at low dose (2.5 microg/g insect) . Injection of chitinase prior to the moult resulted in decreased cuticle thickness . The recombinant proteins caused chronic effects when fed, causing reductions in larval growth and food consumption by up to 60% . The oral toxicity of chitinase was not increased by attaching GNA in the fusion protein, due to degradation in the larval gut, preventing GNA acting as a "carrier".

Gene, 2004 Oct 13, 340(2), 261 - 6
Production and secretion of biologically active recombinant canine growth hormone by Pichia pastoris; Ascacio-Martinez JA et al.; Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Muts) and methanol utilization plus (Mut+) based on Pichia pastoris . Led by the Saccharomyces cerevisiae alpha-mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form . The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 microg/ml for Muts and Mut+ constructs, respectively . As judged by densitometry of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the hormone produced by the fermented Mut(s) strain upon induction with methanol reached 24 microg/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut+ counterpart . Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay.

J Biochem Mol Biol, 2004 May 31, 37(3), 282 - 91
Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris; Xiong AS et al.; Phytases catalyze the release of phosphate from phytic acid . Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid . Two hundred colonies that exhibited potential phytase activity were selected for further study . The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113 . The phytase gene from A . niger 113 (phyI1) was isolated, cloned, and characterized . The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively . The identity between phyI1 and phyA from SK-57 was 89% and 96% . A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P . pastoris biased codon . For the phytase expression and secretion, the construct was integrated into the genome of P . pastoris by homologous recombination . Over-expressing strains were selected and fermented . It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid . The activity of the resulting phytase was 9.5 U/mg . Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species . An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.

Int Arch Allergy Immunol, 2004 Nov, 135(3), 187 - 95 Epub 2004 Nov.
Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests; van Oort E et al.; BACKGROUND: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools . OBJECTIVE: The objective of this study was to investigate whether glycosylation by P . pastoris interferes with the specificity of diagnostic tests . METHODS: An autoantigen involved in Wegener's disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P . pastoris . O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition . Results: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans . We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals . These IgG responses were closely correlated . The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects . The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan . IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis . CONCLUSION: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.

Appl Environ Microbiol, 2004 Oct, 70(10), 6337 - 41
Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422; Poza M et al.; The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames . Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E . coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

Appl Environ Microbiol, 2004 Oct, 70(10), 5905 - 11
Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala; Fredlund E et al.; We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala . In aerobic batch culture, P . anomala grows in the respiratory mode with a high biomass yield (0.59 g {dry weight} of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production . Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production . Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate . No activation of these enzyme activities was observed after a glucose pulse . However, after the shift to oxygen limitation, both enzymes were activated threefold . Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation . Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth . Overall, our results demonstrate that P . anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.

J Mol Biol, 2004 Oct 22, 343(3), 759 - 69
The major human structural IgE epitope of the Brazil nut allergen Ber e 1: a chimaeric and protein microarray approach; Alcocer MJ et al.; A protein microarray system containing different dilutions of 77 related and non-related proteins was used to show that IgE from subjects allergic to Brazil nut specifically recognise the seed 2S albumin protein (Ber e 1) . Further, correctly folded chimaeric 2S albumin proteins containing structural epitope replacement were constructed and directed to the secretion pathway of the methylotropic yeast Pichia pastoris . Through the use of a chimaeric protein microarray system together with sera from a panel of 18 well-characterised Brazil nut allergic subjects, a structural IgE epitope of Ber e 1 was mapped to a helix-loop-helix region . The same structural region has been previously reported as the immunodominant region in related food allergens by different techniques . In conclusion, the combination of chimaeric proteins and protein microarrays will greatly facilitate the screening of a large number of individuals for a particular structural epitope and help to further our understanding of how proteins are recognised by the adaptive immune system.

Wei Sheng Yan Jiu, 2004 Jul, 33(4), 497 - 8
{Identification of types of yeast polluting the foodstuff saled in Beijing}; He Y et al.; OBJECTIVE: To investigate and to specify types of yeasts polluting the foodstuff saled in Beijing market . METHODS: The yeasts strains isolated from the samples delivered was identified by API 20C AUX . Combining the results obtained from API 20C AUX tests and morphological observation of yeasts . RESULTS: There were 29 strains, and 15 species . In the 29 strains, the Pichia membranaefaciens was the mostly prevalent, accounting for 20.69% followed by Zygosaccharomyces bailii (17.24%) and Candida krusei (10.34%) . Totally, there were spoilage yeasts of 7 species 19 strains, accounting for a percentage of 65.52 . Foodstuff made of bean and sauce were the mostly polluted by yeasts . CONCLUSION: Relevant supervision and inspection work should be strengthened.

Prep Biochem Biotechnol, 2004 Aug, 34(3), 239 - 52
Purification and identification of recombinant hirudin and its degradation products expressed in Pichia pastoris; Zhou WB et al.; The purification and identification of recombinant hirudin (r-hirudin) (rHV2-Lys47) and its several C-terminal proteolytic degradation derivatives, produced by Pichia pastoris, were described . The high-purity rHV2-Lys47 of above 99% and its three degradation products were obtained by a straightforward two-step chromatography procedure, a combination of cation exchange and reverse phase chromatography, with a recovery yield of 74% for hirudin . The purified rHV2 had the predicted N-terminal amino acid sequence and the derivatives were the degradation products of hirudin, short of one to three amino acid residues at C-terminal.

World J Gastroenterol, 2004 Nov 1, 10(21), 3188 - 90
Expression of human augmenter of liver regeneration in pichia pastoris yeast and its bioactivity in vitro; Liu Q et al.; AIM: To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro . METHODS: With PCR and gene recombination techniques, cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9 . The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K . The recombinant plasmid pPIC9K-hALR was transformed into GS115 with electroporation . hALR was expressed by GS115 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot . The effects of hALR on in vitro proliferation of QGY and HepG(2) cells were evaluated by (3)H-TdR methods . RESULTS: The correctness and integrity of recombinant plasmids pPIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods, respectively . hALR as a secretive protein was successfully expressed by GS115 . Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid pPIC9K-hALR . The results of Western blot of hALR showed the specific band . The high qualitative hALR was obtained through ultrafiltration . hALR could stimulate in vitro proliferation of QGY and HepG(2) cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG(2) . CONCLUSION: The hALR as a secretive protein can be successfully expressed by GS115 . It may stimulate in vitro proliferation of QGY and HepG(2) cells at a dose-dependent manner . But QGY and HepG(2) cells have different reactivities to hALR.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 9 - 15
Functional importance of Asp37 from a family 11 xylanase in the binding to two proteinaceous xylanase inhibitors from wheat; Tahir TA et al.; Aspergillus niger xylanase is a target enzyme of the two wheat proteinaceous inhibitors, XIP-I and TAXI-I . We previously suggested that the xylanase "thumb" region was XIP-I binding site . Here, we expressed the Asp37Ala mutant in Pichia pastoris and showed that the mutation abolished the enzyme capacity to interact with both inhibitors, suggesting a direct contact at the active site . The mutant pH profile was altered, confirming the key role of Asp37 in determining the pH optima of glycoside hydrolase family 11 . The results are consistent with a competitive inhibition mode and underline the strategic importance of Asp37 in the inhibition mechanism.

Biochim Biophys Acta, 2004 Sep 1, 1701(1-2), 121 - 8
The inhibition specificity of recombinant Penicillium funiculosum xylanase B towards wheat proteinaceous inhibitors; Brutus A et al.; The filamentous fungus Penicillium funiculosum produces a mixture of modular and non-modular xylanases belonging to different glycoside hydrolase (GH) families . In the present study, we heterologously expressed the cDNA encoding GH11 xylanase B (XYNB) and studied the enzymatic properties of the recombinant enzyme . Expression in Escherichia coli led to the partial purification of a glutathione fusion protein from the soluble fraction whereas the recombinant protein produced in Pichia pastoris was successfully purified using a one-step chromatography . Despite O-glycosylation heterogeneity, the purified enzyme efficiently degraded low viscosity xylan {K(m)=40+/-3 g l(-1), V(max)=16.1+/-0.8 micromol xylose min(-1) and k(cat)=5405+/-150 s(-1) at pH 4.2 and 45 degrees C} and medium viscosity xylan {K(m)=34.5+/-3.2 g l(-1), V(max)=14.9+/-1.0 micromol xylose min(-1)k(cat)=4966+/-333 s(-1) at pH 4.2 and 45 degrees C} . XYNB was further tested for its ability to interact with wheat xylanase inhibitors . The xylanase activity of XYNB produced in P . pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K(i) of 89.7+/-8.5 and 2.9+/-0.3 nM, respectively, whereas no inhibition was detected with TAXI-II . Physical interaction of both TAXI-I and XIP-I with XYNB was observed using titration curves across a pH range 3-9.

Biochemistry, 2004 Oct 5, 43(39), 12686 - 91
Pig muscle carnitine palmitoyltransferase I (CPTI beta), with low Km for carnitine and low sensitivity to malonyl-CoA inhibition, has kinetic characteristics similar to those of the rat liver (CPTI alpha) enzyme; Relat J et al.; The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of long-chain fatty acids . There are two well-characterized isotypes of CPTI: CPTIalpha (also known as L-CPTI) and CPTIbeta (also known as M-CPTI) that in human and rat encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition . Kinetic hallmarks of the CPTIalpha are high affinity for carnitine and low sensitivity to malonyl-CoA inhibition, while the opposite characteristics, low affinity for carnitine and high sensitivity to malonyl-CoA, are intrinsic to the CPTIbeta isotype . We have isolated the pig CPTIbeta cDNA which encodes for a protein of 772 amino acids that shares extensive sequence identity with human (88%), rat (85%), and mouse (86%) CPTIbeta, while the degree of homology with the CPTIalpha from human (61%), rat (62%), and mouse (60%) is much lower . However, when expressed in the yeast Pichia pastoris, pig CPTIbeta shows kinetic characteristics similar to those of the CPTIalpha isotype . Thus, the pig CPTIbeta, unlike the corresponding human or rat enzyme, has a high affinity for carnitine (K(m) = 197 microM) and low sensitive to malonyl-CoA inhibition (IC(50) = 906 nM) . Therefore, the recombinant pig CPTIbeta has unique kinetic characteristics, which makes it a useful model to study the structure-function relationship of the CPTI enzymes.

Can J Microbiol, 2004 Jul, 50(7), 489 - 92
Production of ribonucleotides by autolysis of Pichia anomala mutant and some physiological activities; Lee JS et al.; Various mutants of Pichia anomala were isolated by ethyl methanesulfonate (EMS) treatment and UV irradiation through cycloheximide resistance and KCl sensitivity . The selected mutant HA-2 accumulated a higher content of RNA and grew faster than the wild-type strain in yeast extract-malt (YM) broth . Autolysis of the HA-2 mutant at 60 degrees C and pH 7.0 for 6 h was the best condition to obtain maximum yields of 5'-ribonucleotides, inosinic monophosphate (IMP) (6.2 mg/g biomass) and guanylic monophosphate (GMP) (35.5 mg/g biomass) . The yield of adenylic monophosphate (AMP) (7.8 mg/g biomass) was optimal at 60 degrees C at pH 6.5 for 6 h . The inhibitory activity of the angiotensin-converting enzyme and the nitrite-scavenging activity for autolysates of the HA-2 mutant were about 13.0% and 47.0% higher than those of native strain, respectively.

FEMS Yeast Res, 2004 Oct, 5(1), 87 - 95
Three new beetle-associated yeast species in the Pichia guilliermondii clade; Suh SO et al.; New yeasts in the Pichia guilliermondii clade were isolated from the digestive tract of basidiocarp-feeding members of seven families of Coleoptera . A molecular phylogeny and unique traits placed eight isolates in Candida fermentati and three undescribed taxa in the genus Candida . The new species and type strains are C . smithsonii (type strain NRRL Y-27642T), C . athensensis (type strain NRRL Y-27644T), and C . elateridarum (type strain NRRL Y-27647T) . Based on comparison of small-and large-subunit rDNA sequences, C . smithsonii and C . athensensis form a statistically well-supported subclade with P . guilliermondii, C . xestobii, and C . fermentati; C . elateridarum is basal to this subclade.

FEMS Yeast Res, 2004 Oct, 5(1), 81 - 5
Ogataea falcaomoraisii sp . nov., a sporogenous methylotrophic yeast from tree exudates; Morais PB et al.; Thirteen strains of a new ascospore-forming, methanol-assimilating yeast species were isolated from sap exudates of Sclerolobium sp . (carvoeiro) in two forest fragments in the state of Toncantins, Brazil, and from Hymenaea courbaril (guapinol, jatoba) in Guanacaste Province, Costa Rica . Analysis of the sequences of the D1/D2 large-subunit ribosomal DNA showed that the species belongs to the genus Ogataea (syn . Pichia), and it was described as Ogataea falcaomoraisii . The closest relatives are Candida ortonii and C . nemodendra . The type culture is UFMG-T264-1T (= CBS 9814T = NRRL Y-27756).

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 926 - 31
Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris; Acosta-Rivero N et al.; Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid . In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied . Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment . Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers . In addition, the presence of HCcAg species that might represent trimers and multimers was observed . After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules . Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment . These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P . pastoris cells . Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Sep, 20(5), 513 - 6
{Expression of FMDV VP1 protein in Pichia pastoris and its immunological activity in mice}; Jin HL et al.; AIM: To express and identify bovine O type foot and mouth disease virus protein 1 (FMDV VP1) in yeast Pichia pastoris . METHODS: FMDV vp1 gene was cloned into secretory Pichia pastoris expression vector-pSuperY . After being linearized with enzyme digestion, the vector was transformed into Pichia pastoris SMD1168H by electroporation . The transformant was screened by zeocin . Expressed proteins in yeast were analyzed by SDS-PAGE and Western blot and then were used to immunize mice . RESULTS: The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of recombinant yeast contained VP1 protein . The recombinant VP1 protein could elicit similar humoral and cellular immune responses in mice to traditional FMDV killed vaccine . CONCLUSION: FMDV VP1 is expressed successfully in yeast Pichia pastoris, which lays the foundation for further FMDV vaccine research.

J Biol Chem, 2004 Nov 26, 279(48), 49588 - 98 Epub 2004 Nov 26.
Molecular basis of anti-horseradish peroxidase staining in Caenorhabditis elegans; Paschinger K et al.; Cross-reactivity with anti-horseradish peroxidase antiserum is a feature of many glycoproteins from plants and invertebrates; indeed staining with this reagent has been used to track neurons in Drosophila melanogaster and Caenorhabditis elegans . Although in insects the evidence indicates that the cross-reaction results from the presence of core alpha1,3-fucosylated N-glycans, the molecular basis for anti-horseradish peroxidase staining in nematodes has been unresolved to date . By using Western blots of wild-type and mutant C . elegans extracts in conjunction with specific inhibitors, we show that the cross-reaction is due to core alpha1,3-fucosylation . Of the various mutants examined, one with a deletion of the fut-1 (K08F8.3) gene showed no reaction to anti-horseradish peroxidase; the molecular phenotype was rescued by injection of either the K08F8 cosmid or the fut-1 open reading frame under control of the let-858 promoter . Furthermore, expression of fut-1 cDNA in Pichia and insect cells in conjunction with antibody staining, high pressure liquid chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses showed that FUT-1 is a core alpha1,3-fucosyltransferase with an unusual substrate specificity . It is the only core fucosyltransferase in plants and animals described to date that does not require the prior action of N-acetylglucosaminyltransferase I.

Gene, 2004 Sep 15, 339, 79 - 88
Secreted subtilisin gene family in Trichophyton rubrum; Jousson O et al.; Secreted proteases constitute potential virulence factors of dermatophytes . A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum . Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T . rubrum and other fungi revealed a presumed ancestral lineage comprising T . rubrum SUB2 and Aspergillus SUBs . All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events . We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T . rubrum grown in a medium containing soy protein as a sole nitrogen source . Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus . The set of deduced amino acid sequences of T . rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry.

Biochem J, 2005 Jan 15, 385(Pt 2), 511 - 7
Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy; Natalello A et al.; The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O . The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of alpha-helices . We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding . The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity . Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200-900 cm(-1) carbohydrate region . A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein . To confirm that the FTIR absorption in the 1200-900 cm(-1) region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties . Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.

FEMS Microbiol Lett, 2004 Sep 15, 238(2), 359 - 65
Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts; Buzzini P et al.; A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii . KT4561 was precipitated by ethanol and purified by ion-exchange chromatography . The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa . Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h . The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C . glabrata, 0.03 and 0.23 microg/ml for I . orientalis and 1.50 and 2.25 microg/ml for P . guilliermondii . Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics . The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.

Protein Expr Purif, 2004 Oct, 37(2), 336 - 43
Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR; Barral P et al.; Olive pollen is one of the main causes of allergy in Mediterranean countries . Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far . Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris . The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells . The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC . N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein . NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6 . The recombinant form of Ole e 6 expressed in P . pastoris can be employed for structural and biochemical studies.

Protein Expr Purif, 2004 Oct, 37(2), 273 - 8
Expression of tropomyosin from Blattella germanica as a recombinant non-fusion protein in Pichia pastoris and comparison of its IgE reactivity with its native counterpart; Jeong KY et al.; Tropomyosins derived from invertebrates are well-known pan allergens . However, the allergenicities of recombinant tropomyosins are variable . Here, we undertook to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins . Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution, and recombinant tropomyosin was expressed in Pichia pastoris . The allergenicities of the native and recombinant tropomyosins were compared by ELISA inhibition analysis . Native German cockroach tropomyosin showed 18% IgE-binding reactivity to German cockroach sensitized sera . Recombinant tropomyosin was produced without fusion protein and its N-terminus was blocked like that of the native counterpart . The IgE-binding reactivity of the recombinant was found to be comparable to that of native tropomyosin over the concentration range 1-1000 ng/ml by ELISA inhibition testing . Recombinant German cockroach tropomyosin expressed in Pichia pastoris showed better allergenicity than that expressed in Escherichia coli . Other factors in addition to the structural differences of native and recombinant proteins may also influence the IgE reactivities of tropomyosins.

Biochem Biophys Res Commun, 2004 Sep 3, 321(4), 900 - 4
Yeast cytochrome c is a sequence-specific DNA-binding protein; Bhatnagar A et al.; A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c) . Cyt c purified from P . pastoris or Saccharomyces cerevisiae binds to AOX2UAS . Specific point mutations in AOX2UAS abolish cyt c binding . We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.

J Pharmacol Exp Ther, 2005 Jan, 312(1), 127 - 133 Epub 2004 Sep 8.
C75 {4-Methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic Acid} Activates Carnitine Palmitoyltransferase-1 in Isolated Mitochondria and Intact Cells without Displacement of Bound Malonyl CoA; Yang N et al.; Carnitine palmitoyltransferase 1beta (CPT-1beta) is a key regulator of the beta oxidation of long-chain fatty acids in skeletal muscle and therefore a potential therapeutic target for diseases associated with defects in lipid metabolism such as obesity and type 2 diabetes . C75 {4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic acid} is an alpha-methylene-butyrolactone that has been characterized as both an inhibitor of fatty acid synthase and more recently, an activator of CPT-1 (Thupari et al., 2002) . Using human CPT-1beta expressed in the yeast Pichia pastoris, we demonstrate that C75 can activate the skeletal muscle isoform of CPT-1 and overcome inactivation of the enzyme by malonyl CoA, an important physiological repressor of CPT-1, and the malonyl CoA mimetic Ro25-0187 {{5-{2-(naphthalen-2-yloxy)-ethoxy}-thiophen-2-yl}-oxo-acetic acid} . We also show that C75 can activate CPT-1 in intact hepatocytes to levels similar to those achieved with inhibition of acetyl-CoA carboxylase, the enzyme that produces malonyl CoA . Finally, we demonstrate that concentrations of C75 sufficient for activation of CPT-1 do not displace bound malonyl CoA . We conclude that CPT-1 is an activator of human CPT-1beta and other CPT-1 isoforms but that it does not activate CPT-1 through antagonism of malonyl CoA binding.

Eur J Biochem, 2004 Sep, 271(18), 3635 - 45
Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding; Carotti C et al.; Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens . Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis . Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region . In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris . Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag . Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine . Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P . pastoris . Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed . We also showed that Gas1p contains five intrachain disulphide bonds . The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S . cerevisiae . The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant . We found that C74 is an essential residue for the proper folding and maturation of Gas1p .

Appl Environ Microbiol, 2004 Sep, 70(9), 5503 - 10
Cloning and characterization of gluconolactone oxidase of Penicillium cyaneo-fulvum ATCC 10431 and evaluation of its use for production of D-erythorbic acid in recombinant Pichia pastoris; Salusjarvi T et al.; A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced . Peptide sequences were used to isolate a cDNA clone encoding the enzyme . The cloned gene exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases . Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO . No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria . Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis . The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter . In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions . Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-delta-lactone or (in combination with glucose oxidase and catalase) from glucose . We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts.

Appl Environ Microbiol, 2004 Sep, 70(9), 5407 - 14
Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells; Katahira S et al.; Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan . We used a cell surface engineering system based on alpha-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and beta-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface . In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface . These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA . In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S . cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S . cerevisiae . After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g . These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S . cerevisiae strain.

Plant J, 2004 Sep, 39(6), 894 - 904
Purification and cloning of an esterase from the weed black-grass (Alopecurus myosuroides), which bioactivates aryloxyphenoxypropionate herbicides; Cummins I et al.; Carboxyesterases which activate aryloxyphenoxypropionate (AOPP) graminicides to their bioactive herbicidal acids by hydrolysing the respective ester precursors have been identified in black-grass (Alopecurus myosuroides), a problem weed of cereal crops in Northern Europe . The dominant 40 kDa carboxyesterase was purified 1700-fold and identified as a serine hydrolase by affinity labelling with a biotinylated fluorophosphonate suicide substrate . MS-MS sequencing of a peptide digest identified it to be a member of the GDSL family of serine hydrolases . The full-length A . myosuroides hydrolase (Amgdsh1) was cloned by RACE-PCR and expressed in the yeast Pichia pastoris as a secreted enzyme . Expression was associated with activity towards AOPP esters . AmGDSH1 was predicted to be glycosylated and exported to the apoplast in planta . Based on the analysis of related sequences in monocotyledonous plants an alternative classification of the GDSL plant hydrolase superfamily is suggested and their importance in endogenous metabolism and herbicide bioactivation in crops and weeds discussed.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Sep, 17(3), 258 - 61
{Antigencity identification of recombinant hepatitis E virus ORF2 protein expressed in Pichia pastoris}; Tong YP et al.; BACKGROUND: To determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P . pastoris) . METHODS: By using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P . pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E . The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested . RESULTS: The minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120 . No cross-reaction was found with sera from patients with any other types of hepatitis . The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C . The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P . pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli . CONCLUSION: There may be more extensive conformational epitopes in the rHEV ORF2 from P . pastoris . The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Dec, 17(4), 310 - 4
{Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris}; Li PC et al.; OBJECTIVE: Human papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease . L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial . But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries . As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels . Over-expression of HPV6-L1 protein in P . pastoris was the purpose of this study . METHODS: The whole L1 gene with preferred codons for P . pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins . RESULTS: Three clones were found to produce L1 protein which can be identified with Western blot . Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation . Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use . A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography . CONCLUSION: HPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Jun, 18(2), 181 - 5
{Producing human lactoferrin by high-density fermentation recombinant Pichia pastoris}; Ying G et al.; BACKGROUND: To evaluate expression of human lactoferrin gene by high-density fermentation in recombinant Pichia pastoris on the premise of maintaining its biological activities . METHODS: The neutrophil was isolated from human peripheral blood and its total RNA was prepared . Full-length cDNA of human lactoferrin gene was then obtained by RT-PCR, cloned into expression vector pPIC 3.5 K and transformed into Pichia pastoris strain KM71 . With two-layer filter method, the transformants with high-productivity of human lactoferrin were screened out into fed-batch high-density fermentation . And later, the physical, chemical and biological activities of fermentation product were detected preliminarily . RESULTS: The strain p3.5-k-7 with better productivity of human lactoferrin was screened out into fed-batch high-density fermentation . The fermentation lasted nearly for nine days, with A-600 of culture once above 260 and the highest productivity of human lactoferrin being 115 mg/L, 7.67 times the amount of that in shake flask cultivation . CONCLUSION: The authors successfully realized high-density fermentation expression of human lactoferrin gene in recombinant Pichia pastoris.

J Ind Microbiol Biotechnol, 2004 Sep, 31(8), 345 - 52 Epub 2004 Jul 29.
Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran; Liu ZL et al.; Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production . Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation . In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM . The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth . A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128 . It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural . The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion . The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 133 - 7
Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala; Fredlund E et al.; A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw) . At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10-14 microg ml(-1) headspace . At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced . Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 microg ml(-1) . We conclude that ethyl acetate is a major component of the anti-mould activity . The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P . roqueforti.

Biochem Biophys Res Commun, 2004 Sep 17, 322(2), 585 - 92
Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase; Wu CM et al.; Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils . The properties of mature ECP have been well studied but those of the signal peptide of ECP (ECPsp) are not clear . In this study, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris, and human epidermoid carcinoma cell line A431 to study the function of ECPsp . We found that expression of ECPsp chimeric proteins inhibited the growth of E . coli and P . pastoris but not A431 cells . Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum . In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementally expression of hSPP in P . pastoris system rescued the cell growth . Taken together, we have demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition.

Biochemistry, 2004 Aug 31, 43(34), 10965 - 78
Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation; O'Connell KM et al.; A series of compounds derived from a previously identified substrate analogue of copper amine oxidases (CuAOs) (Shepard et al . (2002) Eur . J . Biochem . 269, 3645-3658) has been screened against six different CuAOs with a view to designing potent and selective inhibitors . The substrate analogues investigated were 4-(1-naphthyloxy)-2-butyn-1-amine, 4-(2-methylphenoxy)-2-butyn-1-amine, 4-(3-methylphenoxy)-2-butyn-1-amine, 4-(4-methylphenoxy)-2-butyn-1-amine, and 4-phenoxy-2-butyn-1-amine . These compounds were screened against equine plasma amine oxidase (EPAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), bovine plasma amine oxidase (BPAO), human kidney diamine oxidase (KDAO), and Arthrobacter globiformis amine oxidase (AGAO) to examine the effect of different substituent groups on potency . Despite the similar structures of the 4-aryloxy analogues evaluated, striking differences in potency were observed . In addition, crystal structures of AGAO derivitized with 4-(2-naphthyloxy)-2-butyn-1-amine and 4-(4-methylphenoxy)-2-butyn-1-amine were obtained at a resolution of 1.7 A . The structures reveal a novel and unprecedented reaction mechanism involving covalent attachment of the alpha,beta-unsaturated aldehyde turnover product to the amino group of the reduced 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor . Collectively, the structural and inhibition results support the feasibility of designing selective mechanism-based inhibitors of copper amine oxidases.

Protein Sci, 2004 Sep, 13(9), 2406 - 15
Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E . coli; White J et al.; Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases . In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule . In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification . The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris . Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency . These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic . Crystals of the E . coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Methods Mol Biol, 2004, 278, 17 - 33
Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris; Pickford AR et al.; The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies . It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes . Here, we describe efficient methods for the production of uniformly 13C,15N-labeled proteins in shake flasks and of uniformly 13C,15N-labeled and 2H,13C,15N-labeled proteins in fermenters . We also provide details of two chromatographic procedures, cation exchange and concanavalin A lectin affinity, that have proved useful in purifying P . pastoris-expressed proteins for NMR studies.

Mikrobiologiia, 2004 May-Jun, 73(3), 406 - 15
{Changes in the fine structure of microbial cells induced by chaotropic salts}; Duda VI et al.; The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures . The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change . High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone) . The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged . The spore core became accessible to stains and showed the presence of regions with high and low electron densities . The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 269 - 82
Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II; Bachhawat P et al.; The biosynthetic activity of yeast Pichia etchellsii beta-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides . Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent . Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h . Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction . The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides . A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed . The cellooligosaccharides were separated to purity as confirmed by TLC . The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Appl Microbiol Biotechnol, 2004 Nov, 66(1), 10 - 26 Epub 2004 Nov.
Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass; Klinke HB et al.; An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given . Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation . The resulting hydrolyzsates contain substances inhibitory to fermentation-depending on both the raw material (biomass) and the pre-treatment applied . An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented . Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed . The furans and phenols generally inhibited growth and ethanol production rate (Q(EtOH)) but not the ethanol yields (Y(EtOH)) in Saccharomyces cerevisiae . Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P) . Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed . Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.

Acta Crystallogr D Biol Crystallogr, 1997 Jan, 53(Pt 1), 112 - 3
Crystallization and preliminary X-ray diffraction studies of a novel killer toxin from a halotolerant yeast Pichia farinosa; Kunishima N; A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations . Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.46 A . The low-salt solution provided crystals that belonged to the same space group, with nearly same cell dimensions . Preliminary diffraction studies showed that the intensity distributions are significantly different between the two crystals . Both types of crystals contained either two or three molecules per asymmetric unit . They diffracted X-rays beyond 2.0 A resolution and were stable to X-ray irradiation.

Acta Crystallogr D Biol Crystallogr, 1996 May, 52(Pt 3), 591 - 3
Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis; Wagner UG; Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants . The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A . The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 874 - 5
Crystallization of rat procathepsin B; Sivaraman J; Rat procathepsin B has been expressed in the yeast Pichia pastoris . To facilitate crystallization of the proform two mutations were introduced: Cys29Ser to avoid self-processing and Ser115Ala to eliminate an N-glycosylation site . The recombinant protein was purified and crystallized by vapor diffusion against mother liquor containing 100 mM KSCN, 100 mM phosphate buffer, pH 6.5 and polyethylene glycol (PEG) 3350 as a precipitating agent . Crystal size was increased by multiple macroseeding . At a 16% PEG concentration trigonal crystals were obtained, with the space group P3(1)21 and a = 99.6, c = 141.4 A, gamma = 120 degrees . They diffract to 2.8 A resolution using a rotating-anode source . At a concentration of 11% PEG, rod-shaped crystals were grown . They are monoclinic, space group P2(1), a = 62.8, b = 67.9, c = 100.4 A, beta = 98.2 degrees and diffract to approximately 3.5 A.

Protein Expr Purif, 2004 Sep, 37(1), 61 - 71
Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris; Barbier GG et al.; NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT) . A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1) . S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris . Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h . Gene copy number in genomic DNA of different clones showed direct correlation with the expression level . S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg . Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration . S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)) . The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1 . S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction . Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

Protein Expr Purif, 2004 Sep, 37(1), 39 - 46
High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris; Fierens K et al.; Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11 . In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L) . The rTAXI-I protein was purified from the P . pastoris culture medium using cation exchange and gel filtration chromatographic steps . rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation . The recombinant protein fold was confirmed by circular dichroism spectroscopy . Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0 . rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively . All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not . Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively . The obtained biochemical characteristics of rTAXI-I produced by P . pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli . The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.

Protein Expr Purif, 2004 Sep, 37(1), 18 - 26
An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris; Damasceno LM et al.; The expression of a humanized single-chain variable domain fragment antibody (A33scFv) was optimized for Pichia pastoris with yields exceeding 4 g L(-1) . A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for immunotherapy of colon cancer . P . pastoris with a MutS phenotype was selected to express A33scFv, which was cloned under regulation of the methanol-inducible AOX1 promoter . We report the optimization of A33scFv production by examining methanol concentrations using fermentation technology with an on-line methanol control in fed-batch fermentation of P . pastoris . In addition, we examined the effect of pH on A33scFv production and biomass accumulation during the methanol induction phase . A33scFv production was found to increase with higher methanol concentrations, reaching 4.3 g L(-1) after 72 h induction with 0.5% (v/v) methanol . Protein production was also greatly affected by pH, resulting in higher yields (e.g., 4.88 g L(-1)) at lower pH values . Biomass accumulation did not seem to vary when cells were induced at different pH values, but was greatly affected by lower concentration of methanol . Purification of A33scFv from clarified medium was done using a two-step chromatographic procedure using anion-exchange and hydrophobic interaction chromatography, resulting in 25% recovery and >90% purity . Pure A33scFv was tested for functionality using surface plasmon resonance and showed activity against immobilized A33 antigen . Our results demonstrate that functional A33scFv can be produced in sufficient quantities using P . pastoris for use in further functionality studies and diagnostic applications.

J Ind Microbiol Biotechnol . 2004 Jul 29; {Epub ahead of print}
Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran; Liu ZL et al.; Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production . Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation . In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM . The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth . A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C(6)H(8)O(3) and a molecular weight of 128 . It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural . The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion . The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

Microbiology, 2004 Aug, 150(Pt 8), 2527 - 34
Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine; Santos A et al.; The use of Pichia membranifaciens CYC 1106 killer toxin against Botrytis cinerea was investigated . This strain exerted a broad-specificity killing action against other yeasts and fungi . At pH 4, optimal killer activity was observed at temperatures up to 20 degrees C . At 25 degrees C the toxic effect was reduced to 70% . The killer activity was higher in acidic medium . Above about pH 4.5 activity decreased sharply and was barely noticeable at pH 6 . The killer toxin protein from P . membranifaciens CYC 1106 was purified to electrophoretic homogeneity . SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa . Killer toxin production was stimulated in the presence of non-ionic detergents . The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain of B . cinerea . The symptoms of infection and grey mould observed in Vitis vinifera plants treated with B . cinerea were prevented in the presence of purified P . membranifaciens killer toxin . The results obtained suggest that P . membranifaciens CYC 1106 killer toxin is of potential use in the biocontrol of B . cinerea.

Biochemistry, 2004 Aug 10, 43(31), 10138 - 48
Human pancreatic lipase-related protein 2 is a galactolipase; Sias B et al.; Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail . A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography . Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids) . Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase . In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat . In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase . These residues are not conserved in HPLRP2 . When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts . This may explain why colipase has such weak effects on HPLRP2 . HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films . Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured . The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Plant Mol Biol, 2004 Feb, 54(3), 335 - 52
Isolation and characterization of delta(6)-desaturase, an ELO-like enzyme and delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris; Kajikawa M et al.; The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids . In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase . Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M . polymorpha . Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris . Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.

J Appl Microbiol, 2004, 97(3), 471 - 6
A cost-effective cane molasses medium for enhanced cell-bound phytase production by Pichia anomala; Vohra A et al.; AIM: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala . METHODS AND RESULTS: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media . The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1) . Different cultural parameters were optimized in cane molasses medium in batch fermentation . The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass) . In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium . The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U) . CONCLUSIONS: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium . Furthermore, the production in cane molasses medium is cost-effective . SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced . This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution .

J Biol Chem, 2004 Oct 15, 279(42), 43789 - 98 Epub 2004 Jul 26.
A search for hyperglycosylation signals in yeast glycoproteins; Conde R et al.; N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types . The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation . The selection of substrates for hyperglycosylation poses a theoretical and practical question . To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model . Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it . On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of alpha-1,6-mannoses . The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites . However, a comparison of invertases secreted by S . cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.

FEBS Lett, 2004 Jul 30, 571(1-3), 67 - 73
Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris; You WK et al.; A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots . The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized . The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin . The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo . However, it did not make any considerable alterations on other blood coagulation factors . Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.

Curr Med Chem, 2004 Jul, 11(13), 1793 - 800
Engineered killer mimotopes: new synthetic peptides for antimicrobial therapy; Magliani W et al.; This review deals with a novel approach to produce synthetic antibiotic peptides (killer mimotopes), similar to those described for the conversion of epitopes into peptide mimotopes, allowing their use as surrogate vaccines . Synthetic peptides pertaining to the complementary determining regions (CDRs) of a recombinant antiidiotypic antibody (PaKTscFv), which mimic the wide spectrum of microbicidal activity of a killer toxin produced by the yeast Pichia anomala (PaKT), have proven to act as structural or functional mimotopes of PaKT . This activity appeared to be mediated by interaction with specific cell wall killer toxin receptors (KTRs), mainly constituted by beta glucans . Killer mimotopes have shown in vitro an impressive microbicidal activity against Candida albicans . They were adopted as a model of PaKT- and PaKTscFv-susceptible microorganisms . Optimization through alanine scanning led to the generation of an engineered decapeptide (KP) of a CDR-L1 pertaining antibody fragment with an enhanced in vitro microbicidal activity . It had a potent therapeutic effect against experimental vaginal and systemic candidiasis in normal and immunodeficient mice caused by flucanozole susceptible and resistant yeast isolates . KP exerted a microbicidal activity in vitro against multidrug-resistant eukaryotic and prokaryotic pathogenic microorganisms, which was neutralized by interaction with laminarin (beta 1,3-glucan) . To our knowledge, KP represents the prototype of an engineered peptide fragment derived from a microbicidal recombinant antiidiotypic antibody . It is capable of exerting antimicrobial activity in vitro and a therapeutic effect in vivo presumably acting through interaction with the beta glucan KTR component in the cell walls of pathogenic microorganisms.

Curr Med Chem, 2004 Aug, 11(15), 1983 - 93
Structure and mechanism of monoamine oxidase; Edmondson DE et al.; Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines . A number of mechanism-based inhibitors (MAOI's) have been developed for clinical use as antidepressants and as neuroprotective drugs . To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required . The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three dimensional crystal structures of MAO B (up to 1.7 angstroms resolution) in complex with different reversible (isatin, 1,4-diphenyl-2-butene) and irreversible inhibitors (pargyline, N-(2-aminoethyl)-p-chlorobenzamide, and trans-2-phenylcyclopropylamine) . The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an "entrance" cavity and an "active site" cavity . These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate . The amine function of the bound substrate approaches the re face of the bent and "puckered" covalent FAD through an "aromatic cage" formed by two tyrosine residues that are perpendicular to the plane of the flavin ring . No amino acid residues that could function as acids or bases are found near the catalytic site . The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer mechanism .

Immunol Lett, 2004 Jul 15, 94(3), 253 - 9
Recombinant hydrophilic human gp100: uptake by dendritic cells and stimulation of autologous CD8+ lymphocytes from melanoma patients; Frankenburg S et al.; Native gp100, a glycoprotein highly expressed in the majority of melanomas, contains several immunogenic peptides that are recognized by cytotoxic lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules . The objective of this study was to evaluate the ability of dendritic cells (DCs) from melanoma patients to take up gp100 protein and stimulate specific autologous CTL . The gp100 used in this study was a recombinant molecule with diminished hydrophobicity, HR-gp100, produced in Escherichia coli bacteria and in Pichia pastoris yeast . Stimulation of CD8+ T cells from melanoma patients with HR-gp100-loaded DC was visualized by confocal microscopy using stained target cells, and was quantitatively measured by the production of IFN-gamma using an ELISPOT assay . The results showed that HR-gp100 protein, produced either in bacteria or in yeast, when loaded on DC from melanoma patients, stimulated autologous CD8+ lymphocytes . By direct visualization, these lymphocytes were found in close contact with dead melanoma cells, and to contain membrane material transferred from stained melanoma cells; in cultures containing control lymphocytes stimulated with unloaded DC, no melanoma cell killing was observed . In ELISPOT assays, increased number of IFN-gamma-producing CD8+ T lymphocytes from patients, but not from healthy controls, were measured upon stimulation with HR-gp100-loaded DC . HR-gp100 could represent a useful tool to load DC with multiple immunogenic epitopes/antigen-derived epitopes for the immunotherapy of melanoma.

Biotechnol Lett, 2004 May, 26(10), 803 - 6
Biodegradation of polycyclic aromatic hydrocarbons by Pichia anomala; Pan F et al.; Pichia anomala 2.2540, isolated from soil contaminated by crude oil, degraded naphthalene, dibenzothiophene, phenanthrene and chrysene, both singly and in combination . The yeast degraded 4.5 mg naphthalene l(-1) within 24 h . Phenanthrene was degraded after a lag of 24 h . When a mixture of all four polycyclic aromatic hydrocarbons was treated at either 0.1-1.6 mg l(-1) or 3.1-5.3 mg l(-1), naphthalene was completely degraded first within 24 h, followed by phenanthrene and dibenzothiophene after 48 h . Chrysene, which remained in the mixture even after 96 h, could be degraded along with naphthalene . Chrysene at 0.7 and 1 mg l(-1), in the presence of 4.3 and 65 mg naphthalene l(-1), respectively, was removed within 96 h.

Biotechnol Lett, 2004 Jun, 26(11), 885 - 90
Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae; Wang Y et al.; To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S . cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker . The rDNA results in integration of multiple copies of the target genes . The industrial stain of S . cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S . cerevisiae NAN-127 and NAN-123 . Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S . cerevisiae . Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.

Biotechnol Lett, 2004 Jun, 26(12), 1013 - 7
Improvement of recombinant hirudin production by controlling NH4+ concentration in Pichia pastoris fermentation; Yang J et al.; In recombinant Pichia pastoris fermentation for hirudin production in a 5 l fermenter, a new strategy was explored to match the short fermentation time at low NH4+ concentration with decreased hirudin degradation at high NH4+ concentration . A combination of a defined medium containing initial 0.025 m NH4+ with NH4+ addition up to 0.6 m in the growth phase was achieved in both the improvement of hirudin production and the repression of hirudin degradation . Intact and total hirudin reached 2.63 g l(-1) and 4.25 g l(-1), respectively.

Methods Mol Biol, 2004, 267, 277 - 86
High-throughput expression in microplate format in Pichia pastoris; Bottner M et al.; The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins . To serve the increasing demand for clones expressing different cDNAs, we developed a cultivation and induction protocol amenable to automation to increase the number of clones screened for protein expression . Therefore cDNAs are cloned for intracellular expression . The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins . Expression is controlled by the tightly regulated and highly inducible alcohol oxidase 1 (AOX1) promoter . The screening procedure is based on a culture volume of 2 mL in a 24-well format . Lysis of the cells occurs via chemical lysis without mechanical disruption . Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones that produce heterologous protein with a yield of 2 mg per L culture volume or higher.

Biotechnol Lett, 2004 Jul, 26(14), 1157 - 62
In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris; Cha HJ et al.; The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest . Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2 . Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P . pastoris.

J Agric Food Chem, 2004 Jul 28, 52(15), 4928 - 34
Structural characterization of potato protease inhibitor I (Cv . Bintje) after expression in Pichia pastoris; van den Broek LA et al.; In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating . A cDNA coding for PI-1 from cv . Bintje was cloned and expressed in Pichia pastoris . Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature . The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins . Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit . The transition temperature for the recombinant PI-1 was 88 degrees C . Similar results were obtained for a partially purified pool of native PI-1 from cv . Bintje .

J Struct Funct Genomics, 2004, 5(1-2), 29 - 44
Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics; Prinz B et al.; We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative . We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures . Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis . We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S . cerevisiae and P . pastoris on a scale of 5 to 10 mg . Both batch and fed batch fermentation methods were applied to S . cerevisiae . The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins . We carried out only fed batch fermentations on P . pastoris strains . Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression . The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag . They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography . This was followed by gel filtration for further purification and for buffer exchange . This three-step purification procedure is necessary to obtain highly purified proteins from yeast . The purified proteins have successfully been subjected to crystallization and biophysical analysis.

Biochemistry, 2004 Jul 27, 43(29), 9352 - 60
N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules; Kurniawan ND et al.; Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis . Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported . This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described . In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris . None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy . We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding . Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain . Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average {(1)H}-(15)N heteronuclear NOE value for each of the two domains . These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor . The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

J Ind Microbiol Biotechnol, 2004 Aug, 31(7), 330 - 4 Epub 2004 Jul 15.
Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins; Zhang W et al.; This paper provides an approach for optimizing the cell density (Xc) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon tau (INF-tau) . The objective was to maximize the volumetric productivity (Q, mg INF-tau l(-1) h(-1)), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of Xc and D within the ranges 150< or = Xc < or =450 g cells (wet weight) l(-1) and 0.1 microm< or = D< or =0.9 microm (microm=0.0678 h(-1), the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor) . The methanol and medium feed rates that resulted in the desired Xc and D were determined based on the mass balance . From the RSM model, the optimal Xc and D were 328.9 g l(-1) and 0.0333 h(-1) for a maximum Q of 2.73 mg l(-1) h(-1) . The model of specific production rate (rho, mg INF-tau g(-1) cells h(-1)) was also established and showed the optimal Xc 287.7 g l(-1) and D=0.0361 h(-1) for the maximum rho (predicted to be 8.92 x 10(-3) mg(-1) g(-1) h(-1)) . The methanol specific consumption rate (nu, g methanol g(-1) cells h(-1)) was calculated and shown to be independent of the cell density . The relationship between nu and mu (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain . The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.

Metab Eng, 2004 Jul, 6(3), 229 - 38
Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae; Jin YS et al.; Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes . Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S . cerevisiae, a yeast traditionally used in ethanol production from hexose . However, recombinant S . cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose . To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth . FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding . Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions . However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained . These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.

Biol Chem, 2004 Jun, 385(6), 517 - 24
Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation; Bayes A et al.; Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS . The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244) . Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6 . A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production . Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism . Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.

J Biol Chem, 2004 Sep 10, 279(37), 38658 - 67 Epub 2004 Jul 12.
Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor; Reddy ST et al.; The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome . The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9 . Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR . A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket . To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris . Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate) . Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9 . The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis . Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

Chin Med Sci J, 2004 Jun, 19(2), 78 - 83
Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles; Li HZ et al.; OBJECTIVE: To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg) . METHODS: The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg . The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly . The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization . RESULTS: The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD . It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles . Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system . Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg . CONCLUSION: The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Protein Expr Purif, 2004 Aug, 36(2), 254 - 62
Determination of the complete cDNA sequence, construction of expression systems, and elucidation of fibrinolytic activity for Tapes japonica lysozyme; Takeshita K et al.; The lysozyme of the marine bivalve, Tapes japonica (13.8 kDa), belongs to the invertebrate lysozyme family and displays both chitinase and isopeptidase activities . We determined the complete cDNA sequence and constructed effective expression systems for this enzyme using Escherichia coli (BL21) and Pichia pastoris . The native and recombinant proteins indicated lysozyme activity and isopeptidase activity, including the proteolysis of d-dimer, a plasminolytic product of stabilized polymeric fibrin . These results will be utilized for the structural and functional study of invertebrate lysozymes, and for the development of applications for thrombosis therapies.

Protein Expr Purif, 2004 Aug, 36(2), 165 - 9
Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris; Zhong X et al.; Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters . In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris . The catalytic activity of the recombinant enzyme was assayed . A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form . The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture . Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s) . Our study is the first report on the heterologous expression of tannase suggesting that the P . pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.

Acta Biochim Biophys Sin (Shanghai), 2004 Jul, 36(7), 513 - 7
Expression of recombinant chinese bovine enterokinase catalytic subunit in P . pastoris and its purification and characterization; Fang L et al.; Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins . cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of alpha-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His6 encoding gene by PCR . Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris . Secreted expression of recombinant EKL-His6 was attained by methanol induction and its molecular weight is 43 kD . Because of the existence of His6-tag, EKL-His6 was easily purified from P . pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture . This purified EKL-His6 demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.

Plant Physiol, 2004 Jul, 135(3), 1221 - 30 Epub 2004 Jul 09.
The biosynthesis of D-Galacturonate in plants . functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis; Molhoj M et al.; Pectic cell wall polysaccharides owe their high negative charge to the presence of D-galacturonate, a monosaccharide that appears to be present only in plants and some prokaryotes . UDP-D-galacturonate, the activated form of this sugar, is known to be formed by the 4-epimerization of UDP-D-glucuronate; however, no coding regions for the epimerase catalyzing this reaction have previously been described in plants . To better understand the mechanisms by which precursors for pectin synthesis are produced, we used a bioinformatics approach to identify and functionally express a UDP-D-glucuronate 4-epimerase (GAE1) from Arabidopsis . GAE1 is predicted to be a type II membrane protein that belongs to the family of short-chain dehydrogenases/reductases . The recombinant enzyme expressed in Pichia pastoris established a 1.3:1 equilibrium between UDP-D-galacturonate and UDP-D-glucuronate but did not epimerize UDP-D-Glc or UDP-D-Xyl . Enzyme assays on cell extracts localized total UDP-D-glucuronate 4-epimerase and recombinant GAE1 activity exclusively to the microsomal fractions of Arabidopsis and Pichia, respectively . GAE1 had a pH optimum of 7.6 and an apparent Km of 0.19 mm . The recombinant enzyme was strongly inhibited by UDP-D-Xyl but not by UDP, UDP-D-Glc, or UDP-D-Gal . Analysis of Arabidopsis plants transformed with a GAE1:GUS construct showed expression in all tissues . The Arabidopsis genome contains five GAE1 paralogs, all of which are transcribed and predicted to contain a membrane anchor . This suggests that all of these enzymes are targeted to an endomembrane system such as the Golgi where they may provide UDP-D-galacturonate to glycosyltransferases in pectin synthesis.

Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 730 - 7
Functional expression of the human breast cancer resistance protein in Pichia pastoris; Mao Q et al.; We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein . BCRP expression level in P . pastoris was comparable to that in HEK cells . The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors . Photolabeling of BCRP with 8-azido{alpha-32P}ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP . Vanadate-induced trapping of 8-azido{alpha-32P}ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+ . The Km and Vmax values of BCRP for {3H}E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively . This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.

Chembiochem, 2004 Jul 5, 5(7), 972 - 9
Enhanced fructose oxidase activity in a galactose oxidase variant; Deacon SE et al.; Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides . High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor . Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate . Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290 . In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris . We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model . We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.

Electrophoresis, 2004 Jul, 25(13), 2003 - 9
Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis; Koller A et al.; We report on microbore liquid chromatography (microLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris . The glycopeptides were subject to microLC/electrospray ionization/mass spectrometry (ESI-MS) and microLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man(7)GlcNAc(2) and Man(14)GlcNAc(2) . Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures . For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-N-glycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS) . Excellent separation of the possible positional isomers was attained, suggesting one for Man(9)GlcNAc(2), two for Man(10)GlcNAc(2), three for Man(11)GlcNAc(2), Man(12)GlcNAc(2), and Man(13)GlcNAc(2), and two for Man(14)GlcNAc(2) . The CE results provided complementary information to the microLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.

Drug Metab Dispos, 2004 Oct, 32(10), 1069 - 74 Epub 2004 Jun 30.
Construction of expression system for human alpha1-acid glycoprotein in Pichia pastoris and evaluation of its drug-binding properties; Nishi K et al.; Human alpha1-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands . This is the first report of the recombinant hAGP (rhAGP) . In this study, rhAGP was expressed in the methylotropic yeast Pichia pastoris (GS115) using the expression vector, pPIC9, and then purified by anionic exchange, hydrophobic interaction, and gel filtration chromatography . The molecular weight of rhAGP was much lower than that of hAGP, because of the difference in glycan chain content . Results of glycopeptidase F digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP . The results of circular dichroism spectra measurement indicated that rhAGP predominantly formed a beta-sheet-rich structure that was the same as that of hAGP and typical of the lipocalin family . From the experiments using AGP-binding drugs (chlorpromazine, warfarin, and progesterone) and quinaldine red as a probe for the binding site, it was indicated that rhAGP also had the same ligand-binding capacity and binding site structure as hAGP . These findings strongly suggest that this recombinant hAGP (rhAGP) is very useful for the exploration of the ligand-binding site and biological function of hAGP.

J Biol Chem, 2004 Sep 10, 279(37), 38287 - 93 Epub 2004 Jun 29.
Soluble, oligomeric, and ligand-binding extracellular domain of the human alpha7 acetylcholine receptor expressed in yeast: replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility; Avramopoulou V et al.; The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR . This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates . Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size . A second mutant form, alpha7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein . This second mutant protein was water-soluble, expressed at a moderate level (0.5 +/- 0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies . It also bound (125)I-alpha-bungarotoxin with relatively high affinity (K(d) = 57 nm), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine, or nicotine (K(i) = 0.8 x 10(-7) m, K(i) = 1 x 10(-5) m, and K(i) = 0.9 x 10(-2) m, respectively) . All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties . These results show that alpha7-ECD(C116S,Cys-loop) was expressed in P . pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.

Biochemistry, 2004 Jul 6, 43(26), 8290 - 6
The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine; Zhang H et al.; Recently, mutations in the novel polytopic integral membrane protein PfCRT were shown to cause chloroquine resistance (CQR) in the malarial parasite Plasmodium falciparum . PfCRT is not a member of the well-known family of ABC proteins that have previously been associated with other drug resistance phenomena . Thus, the mechanism(s) whereby mutant PfCRT molecules confer antimalarial drug resistance is (are) unknown . Previously, we succeeded in overexpressing PfCRT to high levels in Pichia pastoris yeast by synthesizing a codon-optimized version of the pfcrt gene . Using purified membranes and inside-out plasma membrane vesicles (ISOV) isolated from strains harboring either wild-type or CQR-associated mutant PfCRT, we now show that under deenergized conditions the PfCRT protein specifically binds the antimalarial drug chloroquine (CQ) with a K(D) near 400 nM but does not measurably bind the related drug quinine (QN) at physiologically relevant concentrations . Transport studies using ISOV show that QN is passively accumulated as expected on the basis of previous measurement of the ISOV DeltapH for the different strains . However, passive accumulation of CQ is lower than expected for ISOV harboring mutant PfCRT, despite higher DeltapH for these ISOV.

Adv Biochem Eng Biotechnol, 2004, 87, 97 - 150
A multi-scale study of industrial fermentation processes and their optimization; Zhang S et al.; In this article problems in multi-scale industrial fermentation processes are discussed . The problems are generated virtually, by using computer simulation on three different scales--the molecular scale (genetics), the cellular scale (metabolic regulation), and the reactor engineering scale . Inter-scale observation and operation are deemed to be crucial in the optimization of bioprocesses . Bioreaction engineering based on metabolic flux analysis and control is further elucidated . Optimization methodology for study of multi-scale problems in a fermentation process, based on correlation of data, and the scale-up technique for regulation of several bioprocess parameters are generalized by investigation of two typical fermentation processes . A novel bioreactor system was designed to monitor mass flux (for example substrates and (by-)products) in a fermentation process . It was successfully applied to the optimization and scale-up of an industrial fermentation process for penicillin, erythromycin, chlortetracyclin, inosine, and guanosine, and for production of recombinant human serum albumin and a malaria vaccine by use of the Pichia expression system . Substantial improvement of industrial fermentation productivity was achieved.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1273 - 8
Structural and functional properties of chicken lysozyme fused serine-rich heptapeptides at the C-terminus; Xu X et al.; Two serine-rich heptapeptides, Ser-Ser-Ser-Lys-Ser-Ser-Ser (S6K) and Ser-Ser-Ser-Ser-Ser-Ser-Ser (S7), were fused to the C-terminus of chicken lysozyme (Lz) by genetic modification to improve the functional properties of lysozyme . The cDNAs of S6K-lysozyme (S6K-Lz) and S7-lysozyme (S7-Lz) were inserted into the expression vector of Pichia pastoris and secreted in yeast cultivation medium . The secretion amounts of S6K-Lz and S7-Lz were about 60% of that of wild-type lysozyme (Wt-Lz) . The CD spectra showed that the conformation of S6K-Lz and S7-Lz was conserved regardless of the attachment of serine-rich peptides . The denaturation curves of S6K-Lz and S7-Lz also showed that the conformational changes were very small . The lytic activity of S6K-Lz and S7-Lz was almost the same as that of Wt-Lz, while the bactericidal activity against Escherichia coli of S6K-Lz and S7-Lz was greatly increased . The acetic acid-urea PAGE of phosphatase-treated S6K-Lz and S7-Lz indicated the possibility of phosphorylation of the fused serine-rich heptapeptides.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1286 - 8 Epub 2004 Jun 22.
Expression, purification, crystallization and preliminary X-ray analysis of alpha-L-arabinofuranosidase B from Aspergillus kawachii; Miyanaga A et al.; Alpha-L-Arabinofuranosidase (EC 3.2.1.55) is one of the hemicellulases that cleave the glycosidic bonds between L-arabinofuranoside side chains and various oligosaccharides . In this study, the first crystallization and preliminary X-ray analysis of alpha-L-arabinofuranosidase B from Aspergillus kawachii IFO4308 (AkAbfB), a family 54 glycoside hydrolase, is described . Recombinant AkAbfB was expressed in Escherichia coli and Pichia pastoris . The native crystals of recombinant AkAbfB produced by P . pastoris belong to the orthorhombic space group P2(1)2(1)2(1) (unit-cell parameters a = 39.5, b = 98.2, c = 144.0 A) and diffracted X-rays to a resolution of 1.82 A.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jul, 20(4), 488 - 90
{Cloning of mouse TLR-2 gene and its expression in Pichia pastroris}; Zhao WZ et al.; AIM: To clone the mouse TLR-2 gene and to express it in Pichia pastroris . METHODS: Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing . The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris . The recombinant Pichia pastroris was confirmed by PCR and RT-PCR . Expressed protein was identified by SDS-PAGE and Western blot . RESULTS: The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned . The homology of the cloned gene to published mTLR-2 gene reached 99.84% . The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully . SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000 . Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody . CONCLUSION: The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.

Poult Sci, 2004 Jun, 83(6), 985 - 9
The effect of phytase enzyme and level on nutrient extraction by broilers; Silversides FG et al.; Three experimental phytase enzyme preparations derived from the same Escherichia coli gene but produced in Saccharomyces cerevisiae (A), Pichia pastoris (B), and Pseudomonas fluorescens (C) were compared with a commercial enzyme preparation by addition to wheat-soybean meal diets fed to broiler chicks . A positive control diet contained sufficient available phosphorus for normal broiler growth and a negative control diet was phosphorus deficient . The 4 enzymes were added to the negative control diet at 3 levels each (150, 450, and 1,250 U/kg), and all diets were pelleted above 80 degrees C . Broiler chicks were fed experimental diets from 4 to 21 d . Chick performance and nutrient digestibility showed that the pel leting process inactivated enzymes A and C and the commercial enzyme . When added to the negative control diet, enzyme B had positive effects on broiler performance and calcium and phosphorus digestibility, and increasing levels of enzyme had greater positive effects . Enzyme B also increased the AME and protein digestibility over those of either control diet . These results suggest that enzyme B was not inactivated by pelleting above 80 degrees C, whereas the other enzymes were.

J Biotechnol, 2004 Jul 1, 111(1), 41 - 50
High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris; Brunel L et al.; Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ) . This enzyme has been shown to belong to a new family of lipases . The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme . The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1) . A transformed P . pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme . The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.

Glycobiology, 2004 Sep, 14(9), 757 - 66 Epub 2004 Jun 09.
Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose; Bobrowicz P et al.; A significant percentage of eukaryotic proteins contain posttranslational modifications, including glycosylation, which are required for biological function . However, the understanding of the structure-function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins . Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins . Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly . The PpALG3 gene encoding Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan(5)GlcNAc(2) human N-glycans . Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P . pastoris strains that synthesize complex GlcNAc(2)Man(3)GlcNAc(2) N-glycans with striking homogeneity . Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and beta-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition . Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure-function relationship of N-glycans.

Mycoses, 2004 Jun, 47(5-6), 231 - 5
Pichia anomala fungaemia in immunocompromised children; Bakir M et al.; Pichia anomala is an emerging yeast causing serious nosocomial infections in newborn and immunocompromised children . We describe nosocomial port catheter infection due to P . anomala in three children who were receiving cancer chemotherapy, bloodstream infection in a preterm infant and in an infant with severe combined immunodeficiency . All patients were treated with amphotericin B . All isolates were susceptible to amphotericin B and fluconazole . No recurrence was observed during follow-up in four of five patients . The common clinical feature in all of our patients was the presence of prior antimicrobial therapy.

Appl Environ Microbiol, 2004 Jun, 70(6), 3370 - 6
Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris; Woo JH et al.; The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation . This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut(+)) under control of the AOX1 promoter . There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P . pastoris and the limited capacity of P . pastoris to secrete the immunotoxin . The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction . Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction . The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature . A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth . The effects of adding glycerol and yeast extract to the methanol feed were synergistic . Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production . The synergy was further enhanced by decreasing the induction temperature from 23 to 15 degrees C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system . This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P . pastoris.

Eur J Biochem, 2004 Jun, 271(12), 2462 - 70
Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris; Sola A et al.; Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling . Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source . For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose . Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae . Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations . Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1) . Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed . Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S . cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1) . This provided a first indication that regulation of central carbon metabolism in P . pastoris and S . cerevisiae might be more similar to each other than to P . stipitis.

Eur J Biochem, 2004 Jun, 271(12), 2370 - 8
Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris; Zani ML et al.; Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G . Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases . A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system . Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase . Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies . Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes . The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3 . K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors . Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.

FEBS Lett, 2004 Jun 4, 567(2-3), 214 - 8
The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase; Altenbach D et al.; Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions . Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit . Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa . If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential.

Protein Expr Purif, 2004 Jul, 36(1), 90 - 9
Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system; Chen YH et al.; Human placental alkaline phosphatase was successfully cloned in the yeast system Pichia pastoris . The recombinant enzyme was over-expressed as a secreted protein in the cultured medium . The enzyme was extremely stable, which resulted in a total recovery of the enzyme activity after the purification process . The purified enzyme preparation was apparently homogeneous as examined by the polyacrylamide gel electrophoresis, analytical gel-permeation chromatography, and analytical ultracentrifugation . The final enzyme preparation showed a purification of 803-fold from the culture medium with a specific activity of 578 U/mg of protein . Fluorescence spectroscopic analyses showed multiple unfolding steps in the urea denaturation process of the homodimeric recombinant enzyme . Extensive conformational change of the enzyme in urea was detected by the analytical ultracentrifugation and the size-exclusive chromatography . The quaternary structure of the enzyme is quite stable . No indication of dissociation was observed after extensive tertiary structural changes.

J Ind Microbiol Biotechnol, 2004 Jun, 31(5), 223 - 8 Epub 2004 Jun 03.
A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements; Terentiev Y et al.; An Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris . The vector harbours a conserved A . adeninivorans-derived 25S rDNA sequence for targeting, the A . adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants . Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene . The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.

J Biol Chem, 2004 Jul 30, 279(31), 32796 - 803 Epub 2004 Jun 02.
Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase; Schulz JM et al.; UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway . Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine . The basis of this differential substrate specificity has remained obscure . Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity . We report here a direct test of this hypothesis . In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE . While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate . All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine . The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis . In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates . In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine . Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

J Biochem Biophys Methods, 2004 Jun 30, 59(3), 241 - 51
A simple method to determine trypsin and chymotrypsin inhibitory activity; Yakoby N et al.; A colorimetric method for serine protease inhibition was modified using N-Acetyl-DL-Phenylalanine beta-Naphthylester (APNE) as the substrate and o-Dianisidine tetrazotized (oD) as the dye . The reaction generated a single peak absorbing at 530 nm for both trypsin and chymotrypsin . Standard curves with increasing enzyme concentrations showed strong linearity . A standard curve for the serine protease inhibitor, Bowman-Birk Inhibitor (BBI), has been made using this modified method . The IC50 for 3 U of trypsin was found to be 33 ng and the IC50 obtained for 3 mU of chymotrypsin was 53 ng . A recombinant BBI (rBBI) gene was constructed, cloned and expressed in the yeast Pichia pastoris . Evaluating samples of rBBI for protease inhibitory activity by the gel activity method failed to quantify the inhibitor amounts, due to high sensitivity for trypsin inhibition and low sensitivity for chymotrypsin inhibition . After development, the results could not be quantified, even to the extent that 1 microl of rBBI could not be detected with chymotrypsin inhibition . Therefore, a modified method for trypsin and chymotrypsin inhibition was used to evaluate the level of rBBI-expression for these same samples . The level of rBBI expression was calculated to be 50-56 ng/microl of media . These amounts fit into the range of values previously obtained by Western blot analysis . This modified method allows us to combine the sensitivity of the gel activity method with the quantification attributes of a Western blot . Thus, the modified method represents a significant improvement in speed, sensitivity and reproducibility over the gel activity method.

Biotechnol Appl Biochem, 2005 Feb, 41(Pt 1), 89 - 95
Recombinant human mast-cell chymase: an improved procedure for expression in Pichia pastoris and purification of the highly active enzyme; Lockhart BE et al.; Human mast-cell chymase (EC 3.4.21.39) is a chymotrypsin-like serine protease that is stored in and released from mast-cell granules . This enzyme has been expressed in Pichia pastoris by homologous recombination of the cDNA coding for the mature active chymase into the Pichia genome . Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor . Methanol induction resulted in the secretion of active chymase into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days . Active enzyme was purified from the culture media with a 22% yield of activity by a simple two-step procedure involving hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin . The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE . The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.

J Agric Food Chem, 2004 Jun 2, 52(11), 3612 - 6
Glycosylation modification improved the characteristics of recombinant chicken cystatin and its application on mackerel surimi; Tzeng SS et al.; The recombinant and glycosylation chicken cystatins were expressed and secreted in the broth of Pichia pastoris X-33 transformant with apparent molecular masses (M) of 14 and 55 kDa, respectively . The glycosylation cystatin (glycocystatin) contained a polysaccharide chain that was composed of 50 DP of mannose residues . Because of the polymannosyl chain, the inhibitory ability in glycocystatin was 90.8% of recombinant cystatin . In addition to freeze-thawing stability, the thermal and pH stabilities as well as the susceptibility of glycocystatin were also enhanced . Both cystatins could improve the mackerel surimi gel by inhibiting the gel softening, which was derived from the hydrolysis of catheptic cysteine proteinases . Despite the additional amount of glycocystatin (8 units), twice that of recombinant cystatin, the 40 and 15% increases in breaking force and deformation of gels were also observed . Accordingly, the surimi gel was further improved by enhancing the stability of chicken cystatin.

Microbiol Res, 2004, 159(1), 11 - 7
Chromium accumulation by living yeast at various environmental conditions; Kaszycki P et al.; Yeast tolerance to Cr (III) and Cr (VI) as well as chromium accumulation potential were shown to depend on treatment time, metal concentration, biomass density and the phase of growth . Kinetic studies as exemplified by Pichia guilliermondii ATCC 201911 revealed a biphasic mode of Cr (III) uptake: a rapid sorption phase was followed by a slow process of accumulation, in which the contribution of the cell-bound Cr fraction increased, while the total cellular Cr level remained constant . Cr (VI) uptake was characterized by a time-dependent increase of total Cr and by a constant fractional contribution of the cell-adsorbed chromium, which suggests that the amount of cell-accumulated Cr also tended to increase over time . The resistance to Cr and metal accumulation levels were substantially elevated for a given strain when cultures were treated at high initial biomass densities (1 mg dry weight/ml) of exponentially proliferating cells . Maximum accumulation capabilities ranged between 4.0 and 13 mg Cr (III)/g dry weight and 2-6.7 mg Cr (VI)/g dry weight . The total cell-accumulated Cr contained 29.3% and 52.3% of organically bound chromium for the treatment of P . guilliermondii with Cr (III) and Cr (VI), respectively . Selected yeast strains, under specified physiological conditions, can be applied for bioremediation of environmental Cr contamination, and might be useful too for attempts to obtain chromium-enriched biomass containing biostabilized and nontoxic Cr forms for nutritional applications.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 May, 19(3), 263 - 5
{Preparation and identification of polyclonal antibody to B cell epitope domain of TRP-1}; Li TH et al.; AIM: To prepare the polyclonal antibody to B cell epitope domain of TRP-1 and apply it to study the immunotherapy of vitiligo and melanoma . METHODS: The fusion protein PRSETA/TRP-1 was expressed in E.coli, the polyclonal antibody was prepared by immunizing a rabbit with the protein . The qulity of the antibody was identified by ELISA and Western-blot . RESULTS: (1)The pRSEA/TRP-1 fusion protein was correctly expressed;(2)the polyclonal antibodies to the B cell eoitope domain of TRP-1 were obtained; (3)the rabbit antiserum containing of polyclonal antibody showed high titer and possessed good binding activity to 6His-TRP1 expressed by Pichia pastoris . CONCLUSION: The polyclonal antibody could be applied to the research on B cell epitope domain of TRP-1.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Mar, 19(2), 130 - 1
{Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris}; Wu YS et al.; AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris . METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k . The constructed plasmid was transformed into yeast SMD1168 by electroporation . The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418 . The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot . RESULTS: The protein was produced at a yield of 50 mg per litre of culture . Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000 . CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.

Commun Agric Appl Biol Sci, 2003, 68(4 Pt A), 17 - 24
Biocontrol of foliar pathogens: mechanisms and application; Elad Y; Biocontrol offers attractive alternatives or supplements to the use of conventional methods for plant disease management . Vast experience has been gained in the biocontrol of plant diseases . Prevention of infection by biocontrol agents or suppression of disease is based on various modes of action . Pathogens are typically affected by certain modes of actions and not by others according to their nature (i.e . biotrophs vs . necrotrophs) . Resistance in the host plant may be induced locally or systemically by either live or dead cells of the biocontrol agent and may affect pathogens of various groups . As some pathogens are negatively affected by lake of nutrients in the infection court, competition for nutrients and space was long recognized as antagonism trait . Antibiosis and hyperparasitism affect pathogens of various groups . Other valid mechanisms are reduction of the saprophytic ability and reducing spore dissemination . Recently it was revealed that restraining of pathogenicity factors of the pathogens, i.e . host hydrolyzing proteins or reactive oxygen species takes place when biocontrol is used . It is likely that several modes of action concomitantly participate in pathogens suppression but the relative importance of each one of them is not clear . Examples of effective prevention of infection in the phyllosphere that rely on multiple modes of action will be demonstrated with Trichodermo harzianum T39 (TRICHODEX), Bacillus mycoides and Pichia guilermondii, a filamentous fungus, bacterium and yeast biocontrol agents, respectively . Several commercial products based on microorganisms have been developed and are starting to penetrate the market . However, large-scale use is still limited because of variability and inconsistency of biocontrol activity . In some cases this may be caused by sensitivity of the biocontrol agents to environmental influences . Ways to overcome biocontrol limitations and to improve its efficacy are i . integration of biocontrol with chemical fungicides on a calendar basis or according to ecological requirements of the biocontrol agents relying on the advise of a decision support system; ii . introduction of two or more biocontrol agents in a mixture, assuming that each one of them has different ecological requirements and/or different modes of action . Implementation of one (or more) of these approaches, using biocontrol preparations mentioned above lowered the variability and increased the consistency of disease suppression . The expected long-term result of the implementation of these suggested strategies is reduced risk of uncontrolled epidemics and increase of confidence of growers in using this non-chemical control measure on a large scale.

Biochemistry, 2004 May 25, 43(20), 6312 - 21
A recombinant C121S mutant of bovine beta-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating; Jayat D et al.; The lipocalin beta-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions . Each monomer contains two disulfide bonds and one cysteine at position 121 (C121) . This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability . We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S) . Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH . The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 degrees C . Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion . Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid pH . We suggest that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.

Exp Appl Acarol, 2004, 32(1-2), 119 - 28
Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris; Boue O et al.; The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris . The recombinant protein has shown to induce protection in cattle against infestations of B . microplus under controlled and production conditions . In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process . The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80% . The process yield was 0.55 g of pure Bm95 protein per liter of culture . The 98% of the primary structure of the recombinant protein was verified by mass spectrometry . Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS . The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P . pastoris.

J Agric Food Chem, 2004 May 19, 52(10), 2997 - 3001
Structure-activity relationships of derivatives of fusapyrone, an antifungal metabolite of Fusarium semitectum; Altomare C et al.; Fusapyrone (1) and deoxyfusapyrone (2) are two 3-substituted-4-hydroxy-6-alkyl-2-pyrones isolated from Fusarium semitectum that have considerable antifungal activity against molds . Because of their low zootoxicity and selective action they are potentially utilizable along with biocontrol yeasts for control of postharvest crop diseases . Seven derivatives of 1 (3 and 5-10) and one derivative of 2 (4) were obtained by chemical modifications of the glycosyl residue, the 2-pyrone ring, the aliphatic chain, or a combination thereof, and a structure-activity correlation study was carried out with regard to their zootoxicity and antifungal activity . Derivatives 7-10, as well as 1, were slightly zootoxic in Artemia salina (brine shrimp) bioassays, whereas pentaacetylation of 1 into 3, 5, and 6 resulted in a strong increase in toxicity . Compound 4, the tetraacetyl derivative of 2, was as toxic as 2 . Because the structural changes of 1 that resulted in an increase of biological activity in A . salina bioassay were those that affected mainly the water solubility of the molecule, it appears that toxicity is related to hydrophobicity . Compounds 1 and 2 showed strong antifungal activity toward Botrytis cinerea, Aspergillus parasiticus, and Penicilliun brevi-compactum (minimum inhibitory concentration at 24 h = 0.78-6.25 microg/mL) . Among derivatives 3-10, only compounds 7, 9, and 10 retained some activity, limited to B . cinerea and at high concentration (25-50 microg/mL) . None of the compounds 1-10 inhibited the growth of the biocontrol yeasts Pichia guilliermondii and Rhodotorula glutinis at the highest concentration tested (50 microg/mL).

Vet Immunol Immunopathol, 2004 Jun, 99(3-4), 153 - 67
The biological effects of five feline IFN-alpha subtypes; Baldwin SL et al.; IFN-alpha has been shown to induce both antiviral and antiproliferative activities in animals . This report describes the biological activity of five recently identified feline IFN-alpha subtypes expressed in the Chinese hamster ovary (CHO) cell line (rfeIFN-alpha1{CHO}, rfeIFN-alpha2{CHO}, rfeIFN-alpha3{CHO}, rfeIFN-alpha5{CHO} and rfeIFN-alpha6{CHO}) and the feIFN-alpha6 subtype expressed in and purified from Pichia pastoris (rfeIFN-alpha6{P . pastoris}) . The rfeIFN-alpha{CHO} subtypes were tested for antiviral activity against either Vesicular stomatitis virus (VSV) or feline calicivirus (FCV) infected feline embryonic fibroblast cell line (AH927) or Crandell feline kidney cell line (CRFK) . Antiviral activity was induced against both VSV and FCV infected AH927 cells and VSV infected CRFK cells by all five of the rfeIFN-alpha{CHO} subtypes and rfeIFN-alpha6{P . pastoris} . In addition, the IFN-alpha inducible Mx gene (associated with antiviral activity) was upregulated in vivo 24 h following treatment with rfeIFN-alpha6{P . pastoris}, compared to baseline levels seen prior to treatment . All of the rfeIFN-alpha{CHO} subtypes and rfeIFN-alpha6{P . pastoris} exhibited antiproliferative activity in the FeT-J cell line (an IL-2 independent feline T-cell line) . Both necrosis and apoptosis were observed in rfeIFN-alpha6{P . pastoris}-treated FeT-J cells . The rfeIFN-alpha3{CHO} subtype consistently exhibited lower antiviral and antiproliferative activity compared to that observed with the other four rfeIFN-alpha{CHO} subtypes . In summary, this paper demonstrates that five previously described feIFN-alpha subtypes induce both antiviral and antiproliferative activities in vitro and are capable of upregulating the feMx gene in vivo .

Protein Expr Purif, 2004 Jun, 35(2), 387 - 96
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells; Blume A et al.; UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals . Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism . This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy . For a detailed understanding of the enzyme, large amounts of the pure active protein are needed . Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells . In all these cell types, the expressed enzyme displayed both epimerase and kinase activities . In E . coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells . In all three cell systems, insoluble protein aggregates were also observed . Purification from E . coli resulted in 100microg/L pure and structurally intact protein . For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein . In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme . However, the easier expression in E . coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.

Protein Expr Purif, 2004 Jun, 35(2), 284 - 92
Recombinant human serum amyloid P component from Pichia pastoris: production and characterization; Boysen S et al.; Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris . SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence . Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag . The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted . Treatment of the cell paste with EDTA increased the yield further by about 30% . The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence . Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa . Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP . r-SAP bound to antibodies produced against h-SAP . Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination . These results indicate that r-SAP produced in P . pastoris has the same biological activity as purified h-SAP.

Protein Expr Purif, 2004 Jun, 35(2), 272 - 5
Purification and characterization of inulinase from Aspergillus niger AF10 expressed in Pichia pastoris; Zhang L et al.; The inuA1 gene encoding an exoinulinase from Aspergillus niger AF10 was expressed in Pichia pastoris, and the recombinant enzyme activity was 316U/ml in a 5L fermentor, with the inulinase protein accounting for 35% of the total protein of fermentation broth . The hydrolysis rate of inulin can reach 92%, with a 25U/g inulin enzyme addition, and 90% of fructose content after 6h . Glucose can significantly inhibit the enzymatic hydrolysis of inulin . This is the first report of glucose inhibition of inulinase-catalyzed hydrolysis.

Biochim Biophys Acta, 2004 May 6, 1698(2), 203 - 12
The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris; Lin J et al.; Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity . We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds . Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites . This internal processing provides useful information on the endoproteolytic activity in P . pastoris . We also show that, similar to many plant allergens, the 2S albumins from soybean are stable to heat and chemical treatments.

Dev Cell, 2004 May, 6(5), 649 - 59
The transitional ER localization mechanism of Pichia pastoris Sec12; Soderholm J et al.; COPII vesicles assemble at ER subdomains called transitional ER (tER) sites, but the mechanism that generates tER sites is unknown . To study tER biogenesis, we analyzed the transmembrane protein Sec12, which initiates COPII vesicle formation . Sec12 is concentrated at discrete tER sites in the budding yeast Pichia pastoris . We find that P . pastoris Sec12 exchanges rapidly between tER sites and the general ER . The tER localization of Sec12 is saturable and is mediated by interaction of the Sec12 cytosolic domain with a partner component . This interaction apparently requires oligomerization of the Sec12 lumenal domain . Redistribution of P . pastoris Sec12 to the general ER does not perturb the localization of downstream tER components, suggesting that Sec12 and other COPII proteins associate with a tER scaffold . These results provide evidence that tER sites form by a network of dynamic associations at the cytosolic face of the ER.

Biochem J, 2004 Aug 15, 382(Pt 1), 67 - 74
The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying alpha1,4-N-acetylgalactosaminyltransferase; Mucha J et al.; Insects express arthro-series glycosphingolipids, which contain an alpha1,4-linked GalNAc residue . To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian alpha1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris . Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcbeta1,4GlcNAcbeta1-R alpha-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal beta-N-acetylgalactosamine residues . NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an alpha1,4-N-acetylgalactosaminyltransferase . We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.

J Biotechnol, 2004 Mar 4, 108(2), 185 - 92
High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris; Peng L et al.; Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum . Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins . In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris . The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied . The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain . When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously . The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant . The specific activity of purified rEK(L) was approximately 9000 u mg(-1) . To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P . pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).

J Immunol, 2004 May 15, 172(10), 6167 - 74
Fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro; Pan W et al.; A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1) . The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and approximately 1 g/L of final product was obtained from a three-step purification process . Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes . PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta) . The chimeric protein induced both anti-MSP1-19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did . Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P . falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution . It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations . Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1-19 and AMA-1(III) of PfCP-2.9 . The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.

Appl Environ Microbiol, 2004 May, 70(5), 2639 - 46
In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris; Vervecken W et al.; The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells . In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues . This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues . Here, we describe engineering of the P . pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans . This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus . The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins . The procedures which we developed allow glycan engineering of any P . pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.

Eur J Biochem, 2004 May, 271(10), 2000 - 11
Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues; Bown DP et al.; Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing . A larval gut cDNA library was screened using probes based on these protein sequences . cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC {Barrett, A . J., Rawlings, N . D . & Woessner, J . F . (1998) Handbook of Proteolytic Enzymes . Academic Press, London.}, but with a novel predicted specificity towards C-terminal acidic residues . This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris . The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation . Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases . The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed . Glutamate carboxypeptidase activity was present in larval gut extract from H . armigera . The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized . The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly . We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.

J Biol Chem, 2004 Jul 9, 279(28), 29023 - 30 Epub 2004 May 03.
The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity; Nigam S et al.; Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade . Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail . We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase . Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3) . 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities . These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase . To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities . The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity . In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers . These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function . This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase . In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.

J Biol Chem, 2004 Jul 2, 279(27), 28182 - 6 Epub 2004 May 03.
Nitrate reductase activity is required for nitrate uptake into fungal but not plant cells; Unkles SE et al.; The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport . It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein . In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate . Uptake assays using the tracer (13)NO(3)(-) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant . In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate . The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 588 - 93
Analysis of recombinant human saposin A expressed by Pichia pastoris; Yamada M et al.; Saposins (SAPs) are small glycoproteins required for activation of sphingolipid hydrolysis by lysosomal enzymes . Four SAPs, SAP-A, -B, -C, and -D, are proteolytically cleaved from a single gene product termed prosaposin . The mature coding sequence of human SAP-A tagged with 6-histidine was expressed in Pichia pastoris and the recombinant protein was purified from the culture supernatant by simple purification steps with an immobilized metal ion affinity column, a Concanavalin A column, and reversed-phase HPLC . Secreted SAP-A contained both glycosylated and nonglycosylated forms . Both forms of SAP-A activated galactocerebroside and 4-methylumbelliferyl beta-d-glucoside hydrolysis by galactocerebrosidase and glucocerebrosidase . SAP-A expressed in P . pastoris should be useful for further structural and functional analysis of this protein.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 959 - 60
Properties of mycelial aggregate-specific lectin of Pleurotus cornucopiae produced in Pichia pastoris; Sumisa F et al.; cDNA of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was expressed in Pichia pastoris, and the expression product was purified and characterized . The product was functional, and the hemagglutinating activity was inhibited most strongly by the addition of N-acetyl-D-galactosamine as was the native lectin . The native lectin is a glycoprotein having five glycosylation recognition signals, and the expression product showed slightly larger molecular mass than that of the native one due to further glycosylation.

Yeast, 2004 Apr 15, 21(5), 445 - 53
Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica; Nakagawa T et al.; In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica . The P . methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%) . In P . methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose . Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium . These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth .

Yeast, 2004 Apr 15, 21(5), 437 - 43
Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii; Bae JH et al.; Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine . We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S . cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P . ciferrii . A positive clone was confirmed by nucleotide sequencing . A syringomycin-E resistance phenotype of a S . cerevisiae sur2-null mutant was complemented by expression of the cloned P . ciferrii SUR2 gene . Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S . cerevisiae SUR2 . .

Biotechnol Bioeng, 2004 May 20, 86(4), 458 - 67
Methanol induction optimization for scFv antibody fragment production in Pichia pastoris; Cunha AE et al.; Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors . Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes . Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P . pastoris was used for this work . Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer . Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot . It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration . This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level . A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables . A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)) . Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate . Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW) . The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required . From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality .

Biotechnol Lett, 2004 Mar, 26(5), 443 - 9
Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification; Murthy TV; Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors . The kinase domain of CHK (amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated . Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation . The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active . A simple plate assay to identify colonies of P . pastoris expressing the recombinant protein is also presented.

Mikrobiol Z, 2004 Jan-Feb, 66(1), 91 - 103
{Yeasts--biosorbents of heavy metals}; Podgorskii VS et al.; The sharp increase of the level of environment pollution by heavy metals caused the increase of interest to the problem of live organisms (including microorganisms) resistance to these metals . Biosorption is one of the mechanisms of microorganisms resistance to heavy metals . Yeasts as biosorbents are of special interest . An analysis of the data from literature have shown that the yeast biomass may be used successfully as biosorption material for such metals as Ag, Au, Cd, Co, Cr, Cu, Ni, Pb, U, Th, Zn . Yeasts of genera Saccharomyces, Candida, Pichia are efficient biosorbents of metals . The sorptional system estimation is based on the classic sorption isotherm obtained in the course of equilibrium experiments and depends on pH, properties of metal ions, biomass concentration, preliminary physical or chemical treatment of the biomass, presence of various organic and inorganic ions and on temperature . The yeast biomass may be obtained using numerous industrial processes, that decreases considerably the biosorbent cost . Most yeasts can sorb a wide range of metals or be strictly specific in respect of only one metal . Special attention would be paid to the cell wall which structure determines sorption proceeding mechanisms . Problems of mechanisms of heavy metal biosorption by microorganisms at molecular level are discussed . The review also deals with the newest developments on improving the biosorption processes in microorganisms, yeast in particular.

Methods Mol Biol, 2004, 265, 219 - 38
Purification and assay of recombinant ADAR proteins expressed in the yeast Pichia pastoris or in Escherichia coli; Ring GM et al.; ADARs are found in Metazoans but are not present in yeasts . We have found that the methanol-utilizing yeast Pichia pastoris can be used to efficiently express enzymatically active epitope-tagged ADARs . We describe plasmid construction and protein expression procedures for producing Drosophila ADAR in this system.ADAR expression in Pichia pastoris uses the methanol-inducible alcohol oxidase AOX1 promoter for induction . A Zeocin resistance gene on the plasmid is used to select high copy number tandem integrations of the plasmid constructs . Preparation of extracts by grinding cultures in liquid nitrogen and purification protocols using 6 x HIS and FLAG epitope tags are described . Procedures for preparing radiolabeled dsRNA and for assaying the non-specific RNA editing activity of ADARs are described.ADARs produced in Escherichia coli are not enzymatically active . We describe expression of the ADAR dsRNA binding domains in E . coli using current versions of the T7 promoter based Studier vectors as well as the purification of the domains.

Wei Sheng Yan Jiu, 2004 Jan, 33(1), 81 - 5
{Expression and identification of cecropin D gene cloned in Pichia pastoris yeast cells}; Fang S et al.; OBJECTIVE: Cecropin D gene was cloned into methanol nutritional vector-pPICZ alpha-A, linked with alpha-factor signal peptide . METHODS: Positive recombinant was gotten through PCR and EcoR I restriction enzyme . The positive recombinant vector was transformed into Pichia pastoris yeast strain GS115 . RESULTS: The positive recombinant yeast velum was fermented and its expressive product was assayed by the method of anti-germ . CONCLUSION: Cecropin D gene was cloned into yeast cell and its expressive products has high activity of killing bacteria, Which could be developed into a new type of anti-bacterial medicine.

Gene, 2004 Apr 14, 330, 39 - 47
The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P . pastoris; Sunga AJ et al.; The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins . We describe the use of a novel selectable marker, the P . pastoris formaldehyde dehydrogenase gene (FLD1) for DNA-mediated transformations of this yeast . The product of the FLD1 gene (Fld1p) is required for growth of P . pastoris on methanol as a carbon source and methylamine as a nitrogen source . In both these C(1) pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide . We show that the FLD1 gene can be used as a marker in transformations of a P . pastoris fld1 host by selection on plates containing methylamine . Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1-based vector by their increased resistance to formaldehyde . We provide the FLD1 selection system in a set of P . pastoris expression vectors that are composed almost entirely of P . pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin . The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non-P . pastoris DNA sequences, particularly bacterial antibiotic resistance genes and replication origins, are considered a potential biological hazard to be avoided.

Allergy, 2004 May, 59(5), 485 - 90
Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment; Cortegano I et al.; BACKGROUND: This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3 . In addition, we present its modulation under polluted environmental conditions . Species of the Cupressaceae family are important because of their high sensitization prevalence . METHODS: Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3 . Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris . RESULTS: Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites . By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104) . This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C . arizonica pollen . Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions . CONCLUSIONS: Cup a 3 is a PR-5 protein derived from C . arizonica pollen . The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.

J Agric Food Chem, 2004 Apr 21, 52(8), 2256 - 61
Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris; Chen JJ et al.; It was shown previously that a bacterially expressed mungbean defensin VrCRP exhibited both antifungal and insecticidal activities . To isolate this protein in a large quantity for its characterization, the defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified . The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum, Pyricularia oryza, Rhizoctonia solani, and Trichophyton rubrum and development of bruchid larva . The protein also inhibits in vitro protein synthesis . These biological activities are similar to that of the bacterially expressed defensin . Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using the mutagenesis approach.

Mol Plant Microbe Interact, 2004 Apr, 17(4), 394 - 403
The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b; Shan W et al.; We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae (Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b . Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by fine-structure genetic mapping . Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein . When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene . This response eventually spread to the entire inoculated plant . In some isolates of P . sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection . In other isolates, such as P6497 (race 2) and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate . Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA . Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497 . The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.

Eukaryot Cell, 2004 Apr, 3(2), 359 - 68
Novel p-type ATPases mediate high-affinity potassium or sodium uptake in fungi; Benito B et al.; Fungi have an absolute requirement for K+, but K+ may be partially replaced by Na+ . Na+ uptake in Ustilago maydis and Pichia sorbitophila was found to exhibit a fast rate, low Km, and apparent independence of the membrane potential . Searches of sequences with similarity to P-type ATPases in databases allowed us to identify three genes in these species, Umacu1, Umacu2, and PsACU1, that could encode P-type ATPases of a novel type . Deletion of the acu1 and acu2 genes proved that they encoded the transporters that mediated the high-affinity Na+ uptake of U . maydis . Heterologous expressions of the Umacu2 gene in K+ transport mutants of Saccharomyces cerevisiae and transport studies in the single and double Deltaacu1 and Deltaacu2 mutants of U . maydis revealed that the acu1 and acu2 genes encode transporters that mediated high-affinity K+ uptake in addition to Na+ uptake . Other fungi also have genes or pseudogenes whose translated sequences show high similarity to the ACU proteins of U . maydis and P . sorbitophila . In the phylogenetic tree of P-type ATPases all the identified ACU ATPases define a new cluster, which shows the lowest divergence with type IIC, animal Na+,K(+)-ATPases . The fungal high-affinity Na+ uptake mediated by ACU ATPases is functionally identical to the uptake that is mediated by some plant HKT transporters.

J Biotechnol, 2004 Apr 8, 109(1-2), 169 - 78
Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation; Invernizzi G et al.; Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques . Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity . The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures . Both proteins were obtained in the culture medium at the concentration of 200 microg/ml . Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium . A preliminary NMR characterisation showed that both proteins were correctly folded.

J Biotechnol, 2004 Apr 8, 109(1-2), 115 - 22
High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles; Lenassi Zupan A et al.; Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein . The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS) . Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine . SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%) . We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase . Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems . As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells . In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.

J Biotechnol, 2004 Apr 8, 109(1-2), 103 - 13
Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter; Resina D et al.; A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source . Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures . PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate . Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels . In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source . Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P . pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P . pastoris.

Carbohydr Res, 2004 Apr 28, 339(6), 1041 - 5
Indiscriminate glycosylation of procarboxypeptidase Y expressed in Pichia pastoris; Maeda H et al.; To obtain large amounts of deglycosylated procarboxypeptidase Y (proCPY), in which all of the N-glycosylation sites were replaced by alanine residue by the point mutation method, an expression system was constructed using Pichia pastoris . The secreted enzyme was characterized by SDS-PAGE, native PAGE, MALDI-TOF mass spectrometry, and dynamic light scattering, and the results indicated heterogeneity . The recombinant proCPY contained 29 mol of glucose per mole of protein in average, according to the carbohydrate analysis by the phenol-sulfuric acid method . A large part of the recombinant enzyme absorbed on a Con A column: even the break-through fraction of the column contained 3 mol of glucose per mole of protein . These carbohydrates were removed by the mild alkaline treatment . Since the entire N-glycosylation site had been destructed in the present expression system, the carbohydrates contained in the recombinant proCPY are concluded to be O-linked ones, which bound indiscriminately to serine and/or threonine residues.

Bioresour Technol, 2004 Jul, 93(3), 249 - 56
Dilute-acid hydrolysis for fermentation of the Bolivian straw material Paja Brava; Sanchez G et al.; Hydrolysis of the straw material Paja Brava, a sturdy grass characteristic for the high plains of Bolivia, was studied in order to find suitable conditions for hydrolysis of the hemicellulose and cellulose parts . Dried Paja Brava material was pre-steamed, impregnated with dilute sulfuric acid (0.5% or 1.0% by wt), and subsequently hydrolyzed in a reactor at temperatures between 170 and 230 degrees C for a reaction time between 3 and 10 min . The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) were found at a temperature of 190 degrees C, and a reaction time of 5-10 min, whereas considerably higher temperatures (230 degrees C) were needed for hydrolysis of cellulose . Fermentability of hemicellulose hydrolyzates was tested using the xylose-fermenting yeast species Pichia stipitis, Candida shehatae and Pachysolen tannophilus . The fermentability of hydrolyzates decreased strongly for hydrolyzates produced at temperatures higher than 200 degrees C .

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 685 - 93
Isolation and characterization of the K5-type yeast killer protein and its homology with an exo-beta-1,3-glucanase; Izgu F et al.; K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column . The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da . The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing . The result of an enzyme immuno assay revealed that it was a glycosylated protein . Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no . AJ222862) of Pichia anomala (strain K) . The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 539 - 57
Ethanol production in immobilized-cell bioreactors from mixed sugar syrups and enzymatic hydrolysates of steam-exploded biomass; De Bari I et al.; We investigated ethanol production from mixed sugar syrups . Hydrolysates were prepared from enzymatic saccharification of steam-pretreated aspen chips . Syrups containing 45 g/L of glucose and 12 g/L of xylose were detoxified through two ion-exchange resins and then fermented with Pichia stipitis and Saccharomyces cerevisiae immobilized in Ca-alginate gel beads . Combinations of different gel fractions in the fermentation volume, amount of yeast cells, and ratios of P . stipitis vs S . cerevisiae within each bead were compared . In the best conditions, by using a total beads volume corresponding to 25% of the working volume, we obtained a yield of 0.39 g(ethanol)/g(initial sugars) . This amount of gel entrapped an initial cell concentration of 6 x 1012cells/L with ratio of S . cerevisiae/P . stipitis of 0.25 g/g . Modified stirred-tank reactors were obtained either by adding marbles or by inserting a perforated metal cylinder, which reduced considerably the rupture of beads while visibly improving oxygenation of the medium.

Plant J, 2004 Apr, 38(1), 27 - 37
Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites; Chanliaud E et al.; Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties . In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan) . Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris . Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase) . Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction . Consequent major changes in cellulose fibril architecture were observed . In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility . On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio . No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET . These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes . Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

J Pharmacol Exp Ther, 2004 Aug, 310(2), 695 - 702 Epub 2004 Mar 29.
a rapid in vitro screening for delivery of peptide-derived peptidase inhibitors as potential drug candidates via epithelial peptide transporters; Foltz M et al.; Targeting drugs or prodrugs to a specific enzyme by simultaneously targeting cell membrane carriers for efficient transport should provide the highest bioavailability along with specificity at the site of action . The peptide transporters PEPT1 and PEPT2 are expressed in a variety of tissues, including the brush-border membranes of epithelial cells of the small intestine and kidney . The transporters accept a wide range of substrates and are therefore good targets for a transporter-mediated drug delivery . Here, we report a screening procedure for peptidomimetic drug candidates combining two independent expression systems: 1) a competition assay in transgenic Pichia pastoris yeast cells expressing either mammalian PEPT1 or PEPT2 for identifying substrate interaction with the transporter binding site; and 2) a Xenopus laevis-based oocyte expression of the peptide transporter for assessing electrogenic transport of drug candidates . Based on the known oral availability and in vivo efficacy of the dipeptidyl peptidase IV (DPIV) inhibitor isoleucine-thiazolidide and its peptide-like structure, we first tested whether this compound is a substrate of epithelial peptide transporters . Additionally, a series of structurally related inhibitors were analyzed for transport . We identified various compounds that serve as substrates of the intestinal peptide transporter PEPT1 . In contrast, none of these DPIV inhibitors showed electrogenic transport by PEPT2, although a variety of the compounds displayed good affinities for competition in peptide uptake in PEPT2-expressing cells, suggesting that they may serve as efficient inhibitors . In conclusion, we have applied an in vitro screening system that predicts efficient intestinal absorption of peptide-derived peptidase inhibitors via PEPT1 in vivo.

Poult Sci, 2004 Mar, 83(3), 421 - 7
The yeast production system in which Escherichia coli phytase is expressed may affect growth performance, bone ash, and nutrient use in broiler chicks; Onyango EM et al.; The efficacy of three Escherichia coli-derived phytase preparations on the performance and nutrient utilization of broiler chicks was evaluated . Two hundred sixteen 7-d-old male broiler chicks were grouped by weight into 6 blocks of 6 cages with 6 birds per cage . Six corn-soybean meal-based diets were randomly assigned to cages within each block . The 6 diets were adequate P, very low P, and low P and contained (g of P/kg of diet) 7.7, 4.0, and 5.1, respectively; and low-P diet plus phytase preparation A, B, or C at 1,000 units/kg of feed . All 3 phytase preparations were produced in different yeast production systems with slightly different glycosylation patterns . Preparation A was produced in Pichia pastoris, B in Schizosaccharomyces pombe, and C in Saccharomyces cerevisiae . The chicks were fed the experimental diets from 8 to 22 d of age . Excreta samples were collected between 17 and 21 d of age . At the end of the study, blood was collected, chicks were killed, and tibiae were removed from 3 birds per cage . Weight gain, feed intake, and feed efficiency among the 3 phytase preparations did not differ, although only phytase A diet outperformed (P < 0.05) the low-P diet in terms of weight gain and feed efficiency . All 3 phytase diets outperformed (P < 0.05) the low-P diet in bone mineral content, density, strength, percentage ash, P retention, and serum P levels . Phytase B diet outperformed the adequate-P diet in bone strength . All 3 preparations increased (P < 0.05) Ca retention with phytase B or C showing a better retention of Ca than phytase A . All 3 phytase preparations showed similar P use as indicated by BW gain and tibia bone characteristics.

Life Sci, 2004 Apr 16, 74(22), 2693 - 705
Expression and functional characterization of recombinant human HDAC1 and HDAC3; Li J et al.; Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation . HDACs are known to play key roles in the regulation of cell proliferation; consequently, inhibition of HDACs has become an interesting approach for anti-cancer therapy . However, expression of mammalian HDACs has proven to be difficult . All attempts to express these HDACs in E.coli, Pichia and baculovirus systems were unsuccessful . Here we present the stable expression of human recombinant His-tagged HDAC1 and HDAC3 in mammalian cells . Full-length human genes for HDAC1 and HDAC3 were cloned into the pcDNA 3.1 vector containing a N-terminal His-tag with an enterokinase cleavage site . Recombinant HDAC enzyme activity was only detected after nickel affinity purification due to high activity of endogenous HDACs; and removal of the His-tag increased activity 2-4 fold . Western blots demonstrated the nickel affinity purified rhHDAC1 preparation also contained endogenous HDAC2 and HDAC3; likewise, rhHDAC3 preparation contained endogenous HDAC1 and HDAC2 . Therefore, the active HDAC preparation is actually a multi-protein and a multi-HDAC containing complex . This provides one explanation for the similar IC50 values exhibited by SAHA and MS-275 against nuclear HDACs and rhHDAC1 and 3 preparations . These results demonstrate that recombinant forms of the HDACs can be over-expressed in mammalian cells, isolated as active multi-protein complexes that contain multiple HDAC enzymes, and caution must be used when determining HDAC inhibitor in vitro selectivity.

Insect Biochem Mol Biol, 2004 Apr, 34(4), 305 - 20
Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris; Bown DP et al.; Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize . A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L . A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency . Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes . Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris . The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin . The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2 . This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates . The enzymes showed similar proteolytic activity towards peptide substrates.

J Gen Virol, 2004 Apr, 85(Pt 4), 1049 - 53
Heterologous expression of the modified coat protein of Cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function; Brumfield S et al.; We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification . Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host . We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P . pastoris system assemble into viral protein cages with altered architectures and function . This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages . Heterologous expression in P . pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

J Insect Physiol, 2004 Jan, 50(1), 61 - 71
Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion; Fitches E et al.; The mannose-specific snowdrop lectin (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph . The ability of GNA to act as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1: SFI1) to the haemolymph of lepidopteran larvae was investigated . Constructs encoding SFI1 and an SFI1/GNA fusion protein were expressed in Pichia pastoris . The insecticidal activity of purified recombinant proteins on injection was found to be comparable to published values for SfI1 purified from spider venom {Toxicon 40 (2002) 125} . Whereas neither GNA nor SFI1 alone showed acute toxicity when fed to larvae of tomato moth (Lacanobia oleracea), feeding SFI1/GNA fusion at 2.5% of dietary proteins was insecticidal to first stadium larvae, causing 100% mortality after 6 days . The protein also showed a significant, dose dependent, toxicity towards fourth and fifth stadium larvae, with growth reduced by up to approximately 90% over a 4-day assay period compared to controls . Delivery of intact SFI1/GNA to the haemolymph in these insects was shown by western blotting; haemolymph samples from fusion-fed larvae contained a GNA-immunoreactive protein of the same molecular weight as the SFI1/GNA fusion . SFI1/GNA and similar fusion proteins offer a novel and effective approach for delivering haemolymph active toxins by oral administration, which could be used in crop protection by expression in transgenic plants.

Glycobiology, 2004 May, 14(5), 457 - 66 Epub 2004 Mar 19.
Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin; Bencurova M et al.; Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins . To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin . Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated . Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue . These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA . Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms . Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes . In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue . The mannose-deficient glycoform was a poorer inhibitor for both antibodies . Terminal GlcNAc residues prevented antibody binding . Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only . Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants . Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.

Glycobiology, 2004 May, 14(5), 399 - 407 Epub 2004 Mar 19.
Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P . pastoris; Davidson RC et al.; N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum . Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi . We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP . Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment . However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) were also observed that were recalcitrant to an array of mannosidase digests . These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts . Finally, analysis of the P . pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p alpha-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S . cerevisiae counterpart.

Plant Cell, 2004 Apr, 16(4), 874 - 86 Epub 2004 Mar 12.
Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding; Johansson P et al.; Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism . Thus, XET is a key enzyme in all plant processes that require cell wall remodeling . To provide a basis for detailed structure-function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-A resolution . Even though the overall structure of PttXET16A is a curved beta-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7 . In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional beta-strand and a short alpha-helix . The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation . Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

Hum Reprod, 2004 Apr, 19(4), 849 - 58 Epub 2004 Mar 11.
The recombinant subdomain IIIB of human serum albumin displays activity of gonadotrophin surge-attenuating factor; Tavoulari S et al.; BACKGROUND: Gonadotrophin surge-attenuating factor (GnSAF) is an as yet unidentified ovarian factor that acts on the pituitary to attenuate the pre-ovulatory LH surge . In a previous study, GnSAF bioactivity was proposed to derive, at least in part, from a C-terminal domain (95peptide) of human serum albumin (HSA) . METHODS AND RESULTS: We employ here the expression-secretion system of Pichia pastoris to produce and assay selected recombinant polypeptides of HSA for GnSAF activity . We show that the C-terminal 95peptide of HSA (residues 490-585; subdomain IIIB) can be expressed from P.pastoris in secreted form and supernatants from clones expressing this polypeptide reduce the GnRH-induced LH secretion of primary rat pituitary cultures by 50-82% . When expressed in the same system, HSA domain III (residues 381-585) or full-length HSA (residues 1-585) are inactive . The bioactive subdomain IIIB is also separable from either domain III or full-length HSA on Blue Sepharose chromatography . CONCLUSIONS: Taken together, the findings highlight the putative importance of HSA subdomain IIIB as a GnSAF-bioactive entity and introduce a unique experimental tool to engineer this molecule for structure-function analysis.

J Microbiol Methods, 2004 Apr, 57(1), 33 - 9
A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance; Wan H et al.; Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation . Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin . This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum . In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P . pastoris genome confers resistance to elevated concentrations of soraphen A . Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain . As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.

Biotechnol Bioeng, 2004 Mar 30, 85(7), 761 - 9
Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris; Masuda T et al.; Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris . The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal . Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule . The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule . Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II . These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin .

Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 976 - 83
Glycosylation of onconase increases its conformational stability and toxicity for cancer cells; Kim BM et al.; Onconase (ONC) is currently in Phase III clinical trials as a cancer chemotherapeutic agent . Despite the finding that ONC contains an N-linked glycosylation site (-N69-V70-T71-), only the non-glycosylated form of the protein has been identified to date . We employed the Pichia pastoris expression system to produce recombinant glycosylated ONC (gONC) protein . Approximately 10 mg of ONC protein was secreted per liter of culture media, of which about 80% was glycosylated at N69 . CD spectroscopic analyses revealed that the secondary structure of gONC is identical to that of ONC . We found that gONC contains a high-mannose core structure . Importantly, glycosylation of ONC at N69 greatly increased its toxicity for K-562 cancer cells . Specifically, the IC50 value of gONC was 50-fold lower than that of ONC . Glycosylation increased both the Tm of ONC and its resistance to proteinase K, suggesting that the elevated cytotoxicity of gONC is related to higher conformational stability.

Arch Biochem Biophys, 2004 Jan 15, 421(2), 260 - 6
Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein; Debski J et al.; The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues . It contains four Cys residues forming two disulfide bridges . In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G . mellonella was determined, using electrospray mass spectrometry . The MS analysis was performed on tryptic digests of JHBP . Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195 . Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated . This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.

Protein Eng, 2003 Dec, 16(12), 1139 - 45
Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization; Musidlowska-Persson A et al.; It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE) . The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE) . By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants . All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate . Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa . All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C . Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.

Biosci Biotechnol Biochem, 2004 Feb, 68(2), 376 - 83
Structural and functional characterization of ovotransferrin produced by Pichia pastoris; Mizutani K et al.; Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin . We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system . The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly . The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l . The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein . The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein . Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms . The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.

J Med Food, 2003 Winter, 6(4), 317 - 22
In vitro anti-cancer activities in Caco-2 and HCT-116 cells of recombinant cystatin C prepared by a Pichia expression system; Ogawa M et al.; Caco-2 and HCT-116 cells were used to access growth-inhibition and anti-invasion activity of recombinant cystatin C expressed in Pichia pastoris X33, G12W/H86V . The mutant G12W/H86V prepared by a pilot plant production system showed more than 10% growth inhibition of Caco-2 cells at 0.56-56 nM concentrations . Growth-inhibited cells had lower cathepsin L activity than the control cells that were not treated with the inhibitor . Conversely, the cathepsin B activity was not changed by treatment with G12W/H86V . The in vitro anti-invasion test using HCT-116 cells showed that G12W/H86V suppressed the cell invasion by 15%, while its wild-type cystatin, aspartic protease inhibitor pepstatin A, and matrix metalloproteinase (MMP) inhibitor MMP-2/MMP-9 inhibitor III did not suppress cell invasion . These results indicate that the recombinant cystatin C with higher protease inhibitory activity effectively retards the growth and invasiveness of human colon carcinoma cells.

J Infect Dis, 2004 Mar 1, 189(5), 919 - 29 Epub 2004 Feb 06.
Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma-secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum; Goud GN et al.; cDNAs encoding 2 Ancylostoma-secreted proteins (ASPs), Ancylostoma ceylanicum (Ay)-ASP-1 and Ay-ASP-2, were cloned from infective third-stage larvae (L3) of the hookworm A . ceylanicum and were expressed as soluble recombinant fusion proteins secreted by the yeast Pichia pastoris . The recombinant fusion proteins were purified, adjuvant formulated, and injected intramuscularly into hamsters . Hamsters vaccinated either by oral vaccination with irradiated L3 (irL3) or by injections of the adjuvants alone served as positive and negative controls, respectively . Anti-ASP-1 and anti-ASP-2 antibody titers exceeded 1 : 100000 . Each vaccinated hamster was challenged orally with 100 L3 . Two groups of vaccinated hamsters (i.e., those vaccinated with either irL3 or ASP-2 formulated with Quil A) exhibited significant reductions in adult hookworm burdens, compared with control hamsters . The hookworms recovered from the hamsters vaccinated with ASP-2 plus Quil A were reduced in length . Splenomegaly, which was observed in control hamsters, was not seen in hamsters vaccinated with either irL3 or ASP-2 formulated with Quil A . These results indicate that ASP-2 is a promising molecule for the development of a hookworm vaccine.

Int Immunopharmacol, 2004 Jan, 4(1), 139 - 48
Expression of the negative co-stimulatory ligand sCD152 in the yeast, Pichia pastoris, and its regulation of antigen specific immune responses; Zhu N et al.; CD152, a ligand expressed on the surface of the activated T cells that inhibits co-stimulatory signals, is associated with negative regulation of T-cell activation in the antigen-presenting process . In order to interfere with immune signal transmission, obtain functional proteins with specific immunosuppressive effects, and regulate the immune response, we cloned the full-length extracellular domain of CD152 into the expression vector pPICTLA and transformed Pichia pastoris GS115 cells by electroporation . Yeast colonies expressing and secreting large quantities of the extracellular soluble fragment of CD152 (sCD152) were isolated, and the protein was purified and used in assays designed to investigate the ability of sCD152 to regulate the immune system . In a series of antigen specific immune response interference tests in mice, human allogeneic mixed lymphocyte reaction and antigen specific lymphocyte transformation tests, we found that sCD152 had a marked immunosuppressive effect . This protein reduced antibody titer in mice immunized with BSA, significantly inhibited the killing of antibody-dependent cytotoxicity and CTL-mediated donor target cells, and reduced the expression of IL2 in mice . In the absence of exposure of sCD152, long-term stimulation (>5 days) by the same antigen in vivo partially restored the immune response-correlated stimulation index (SI), showing the characteristics of a primary immune response . Following the deletion of sCD152, however, there was no obvious inhibitory effect on the primary immune response triggered by the nonrelevant antigen OVA, indicating that sCD152 had an antigen-specific immunosuppressive effect and that the inhibitory effect of sCD152 required the co-administration of antigen . These findings suggest that sCD152 may have clinical potential as an immunosuppressive agent.

Yeast, 2004 Feb, 21(3), 249 - 63
Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone; Laborde C et al.; Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector . Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones . Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene . Highest expression was observed with the single-copy clone S27 (250 mg/l) . The modifications of culture conditions in batch and fed-batch culture increase the yield of protein . The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l . Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones . At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3% . Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate . We characterized the recombinant leptin produced by clone S27 . It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site . Moreover, it was found to be biologically active in vitro . The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes .

J Lab Clin Med, 2004 Feb, 143(2), 115 - 24
A covalently linked recombinant albumin dimer is more rapidly cleared in vivo than are wild-type and mutant C34A albumin; McCurdy TR et al.; Mammalian albumins are abundant plasma proteins that exhibit a relatively slow terminal clearance . For this reason they have been fused to potentially therapeutic proteins with rapid terminal clearance to produce fusion proteins with more desirable clearance profiles . A disulfide-linked albumin dimer has been described, but its abundance and stability in plasma are uncertain . To determine whether an obligatory albumin dimer incapable of dissociation would clear less rapidly than monomeric albumin, we expressed 3 recombinant rabbit serum albumin (RSA) polypeptides: H6RSA, RSA modified by the addition of an N-terminal hexahistidinyl tag; H6RSA(C34A), H6RSA with a single cysteine (Cys) 34-to-alanine (Ala) substitution (C34A); and DiRSA, H6RSA(C34A) joined by way of its C-terminus to RSA(C34A) through an intervening hexaglycine spacer . The C34A mutation was introduced to eliminate the possibility of disulfide bond-mediated dimerization . We expressed the proteins with the use of the yeast Pichia pastoris and purified them using nickel-chelate, ion exchange, and gel-filtration chromatography . After radioiodination and injection into rabbits, H6RSA and H6RSA(C34A) exhibited indistinguishable terminal catabolic half-lives (4.9 +/- 0.7 and 4.8 +/- 0.5 days, mean +/- SD), whereas that of DiRSA was reduced to 3.0 +/- 0.3 days (p<.05) . The three proteins circulated in intact form, and their distributions in liver, lung, kidney, heart, and spleen did not differ 24 hours after injection . Although more DiRSA than H6RSA(C34A) was present in urine, in both cases it was in acid-soluble form . Ethyl palmitate treatment reduced the relative acceleration of DiRSA clearance compared with that of H6RSA(C34A), suggesting a role for the reticuloendothelial system in the differential clearance of the larger protein . Our results suggest that an albumin fusion protein should include only a single copy of albumin; that if the fusion protein exceeds a certain size, it may not acquire the slow clearance profile of native albumin; and that albumin dimerization through Cys34 probably does not contribute substantially to albumin metabolism in vivo.

Protein Expr Purif, 2003 Dec, 32(2), 175 - 84
Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris; Gadkari R et al.; The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction . These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit . The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins . The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants . In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system . A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity . Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active . The preliminary data also suggested that P . pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated . This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.

Protein Expr Purif, 2003 Dec, 32(2), 167 - 74
Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris; Zocchi A et al.; A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris . The coding sequence for recGAvi was de novo synthesized based on the codon usage of P . pastoris . RecGAvi is secreted at approximately 330mg/L of culture supernatant . RecGAvi monomer displays a molecular weight of 16.5kDa, as assessed by ESI mass spectrometry . N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4 . The data presented here demonstrate that the P . pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.

Mar Biotechnol (NY), 2001 Mar, 3(2), 126 - 32
Screening and characterization of fructosyl-valine-utilizing marine microorganisms; Sode K et al.; We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast . Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source . Among these strains, a yeast strain identified as Pichia sp . N1-1, produced a Fru-Val-oxidizing enzyme . The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H(2)O(2) with the oxidation of Fru-Val . By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase . This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast.

J Biochem (Tokyo), 2003 Dec, 134(6), 911 - 7
pPIC9-Fc: a vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris as detection reagents in vitro; Liu J et al.; Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates . For ScFv fragments, there is no such universal system available up to now . A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9 . A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc . Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography . The expression yield can reach 10-30 mg/liter of culture medium . The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins . Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.

J Biochem (Tokyo), 2003 Dec, 134(6), 813 - 7
Expression, purification, and characterization of humanized anti-HBs Fab fragment; Ning D et al.; Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV . Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris . This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells . The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter . The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions . The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography . The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg) . The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.

Receptors Channels, 2004, 10(1), 37 - 50
Production of the human D2S receptor in the methylotrophic yeast P . pastoris; Grunewald S et al.; In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D(2S) receptor protein, we have expressed the unmodified D(2S) receptor and various D(2S) receptor fusion constructs under the transcriptional control of the highly inducible promotor of the P . pastoris alcoholoxidase 1 gene in strain SMD1163 . Fusion of the D(2S) receptor gene to the alpha-factor preprosequence proved to be essential for receptor production . For the receptor fusion constructs a gene dosage of more than two copies per cell increased production levels three- to sixfold . Adding various dopaminergic ligands to the induction medium increased yields up to tenfold, reaching 51,500 +/- 5700 receptors/cell . Immunoblot analysis of the effect of tunicamycin on D(2S) receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D(2S) receptor fusion proteins revealed that the high-mannose-type glycosylation of the D(2S) receptor prevents cleavage of the alpha-factor prosequence by the Kex2 endopeptidase . Abolishing glycosylation restored correct processing . Immunogold electron microscopy showed that recombinant yeast cells overproducing the D(2S) receptor developed membrane stacks harboring the receptor protein . The pharmacological profile of the recombinant D(2S) receptor was similar to that reported for neuronal D(2) receptors independent of glycosylation and processing . In conclusion, the D(2S) receptor can readily be produced in P . pastoris with high yield suitable for receptor purification and future structural studies.

Appl Environ Microbiol, 2004 Feb, 70(2), 961 - 6
Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; Eiden-Plach A et al.; Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins . Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin . In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters . In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium . In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin . Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Protein Expr Purif, 2004 Mar, 34(1), 147 - 57
Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization; Wendeler M et al.; The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome . It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase . Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis . With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris . For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter . Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus . Remarkably, the expression of this membrane-active protein in P . pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein . A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant . MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein . Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied . A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy . Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Protein Expr Purif, 2004 Mar, 34(1), 134 - 41
Heterologous expression of maize (Zea mays L.) Mir1 cysteine proteinase in eukaryotic and prokaryotic expression systems; Pechan T et al.; Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1 . A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity . Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity . Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

Biochem Biophys Res Commun, 2004 Mar 5, 315(2), 247 - 54
The monotopic membrane protein human oxidosqualene cyclase is active as monomer; Ruf A et al.; The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals . Therefore, it is an important target for the development of new hypocholesterolemic drugs . Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris . The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes . The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form . Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein . The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.

World J Gastroenterol, 2004 Feb 1, 10(3), 342 - 7
Cloning and expression of human colon mast cell carboxypeptidase; Chen ZQ et al.; AIM: To clone and express the human colon mast cell carboxypeptidase (MC-CP) gene . METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR) . The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to construct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP . The recombinant fusion protein expressed in E . coli was induced with IPTG and purified by amylose affinity chromatography . After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography . The recombinant hMC-CP expressed in Pichia pastoris (P . pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid sequencing and enzyme assay . RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA . The recombinant hMC-CP protein expressed in E . coli was purified with amylose affinity chromatography and heparin agarose chromatography . SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E . coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5) . The N-terminal amino acid sequence confirmed further the product was hMC-CP . E . coli generated hMC-CP showed a very low level of enzymatic activity, but P . pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine . CONCLUSION: The cDNA encoding human colon mast cell carboxypeptidase can be successfully cloned and expressed in E . coli and P . pastoris, which will contribute greatly to the functional study on hMC-CP.

Yeast, 2004 Jan 30, 21(2), 151 - 62
Ion channel activity by Pichia membranifaciens killer toxin; Santos A et al.; The cytocidal effect of Pichia membranifaciens killer toxin on Candida boidinii cells was studied . The halotolerant yeast P . membranifaciens CYC 1106 produces a unique 18 kDa killer toxin that exerts its killer activity against C . boidinii IGC 3430 only in the presence of NaCl . Metabolic events associated with the loss of C . boidinii IGC 3430 viability were quantitatively identical to those known to occur with K1 killer toxin-treated sensitive strains of Saccharomyces cerevisiae . The death of sensitive cells was characterized by a leakage of potassium, an influx of sodium and a decrease in intracellular pH . These effects occurred prior to and concomitantly with cell death, indicating that they were primary effects of the action of the toxin . Here we report that this protein forms ion-permeable channels in liposome membranes . These channels are freely permeable to common physiological ions . We suggest that channel formation is the cytotoxic mechanism of action of P . membranifaciens killer toxin . The channels described here are sufficiently non-selective to mediate cell death through a discharge of cellular membrane potential and changes in ionic homeostasis . No specific effects against killer toxin-treated sensitive cells were observed when the cell cycle was analysed .

Yeast, 2004 Jan 30, 21(2), 141 - 50
Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae; Traff-Bjerre KL et al.; Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S . cerevisiae . However, xylitol is a major by-product . An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S . cerevisiae strains to investigate its effect on xylose utilization . In a recombinant S . cerevisiae strain producing only xylitol dehydrogenase (XDH) from P . stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol . When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively . When the GRE3 gene was deleted in the recombinant xylose-fermenting S . cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P . stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture . Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion .

Biotechnol Bioeng, 2004 Feb 20, 85(4), 367 - 75
Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris; Hohenblum H et al.; The expression of heterologous proteins may exert severe stress on the host cells at different levels . Depending on the specific features of the product, different steps may be rate-limiting . For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described . During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified . Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers . With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression . To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter . An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells . A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source .

J Biol Chem, 2004 Apr 23, 279(17), 17038 - 46 Epub 2004 Jan 30.
Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells; Collins PR et al.; The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated . Only the fully processed and active mature enzyme can be detected in medium in which adult F . hepatica are cultured . However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut . These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen . Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form . Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids . An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess . However, {Gly(26)}procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus . This exogenous processing occurred without the formation of a 30-kDa intermediate form . The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.

Biotechnol Bioeng, 2004 Feb 5, 85(3), 330 - 9
Expression of recombinant vitellogenin in the yeast Pichia pastoris; Ding JL et al.; Vitellogenin (Vtg) plays vital roles as precursor to the yolk proteins and as carrier for lipids, carbohydrates, phosphates, metal ions, vitamins, and hormones into the oocytes during the massive deposition of yolk nutrients for subsequent nourishment of the developing embryos . Reproductive success is highly sensitive to the nutritional quality of the broodstock diet, which greatly affects the egg and larval viability . We present a novel strategy for genetically engineering a Pichia pastoris yeast strain that constitutively produces recombinant Vtg (rVtg), for application as an enriched feed . The tilapia Oreochromis aureus Vtg (OaVtg) cDNA (5.3 kb) was cloned into a nonsecretory pGAPZA vector . Clones containing up to 31 copies of glyceraldehyde-3-phosphate dehydrogenase (GAP)-promoter-driven Vtg expression cassettes were isolated . These clones expressed a membrane-associated intracellular rVtg protein of 194 kDa, constituting up to 1.16% of total protein . To facilitate future purification of rVtg, we explored the possibility of secreting rVtg using the native Vtg secretion signal and the alpha-factor secretion signal of Saccharomyces cerevisiae . However, neither signal promoted the secretion of rVtg . The clones maximally expressed rVtg at 23 degrees C, reaching a peak at 22 h in shake flasks and 16 h in a fermentor . The clones exhibited a significant increase in essential amino acids and long-chain polyunsaturated fatty acids, which are important for its application as a high-quality nutrient feed .

Infect Immun, 2004 Feb, 72(2), 709 - 16
Binding and agglutination of Streptococcus pneumoniae by human surfactant protein D (SP-D) vary between strains, but SP-D fails to enhance killing by neutrophils; Jounblat R et al.; Recombinant human surfactant protein D (SP-D) expressed in Escherichia coli, consisting of the head and neck regions of the native molecule, bound to all strains of Streptococcus pneumoniae that were tested, but the extent of binding varied between strains of differing capsular serotypes . The recombinant protein expressed in the yeast Pichia pastoris did not bind . Full-length native SP-D aggregated pneumococci in a calcium-dependent manner that was inhibited by maltose acting as a competitive sugar . The ability of SP-D to modulate the uptake and killing of pneumococci by human neutrophils was also addressed . Neither recombinant truncated SP-D nor native full-length SP-D enhanced the killing of pneumococci by human neutrophils . Aggregation of pneumococci varied not only between strains of the same multilocus sequence type and different serotypes but also between strains of the same serotype . However, use of recombinant strains in which the serotype had been changed showed that the degree of aggregation was influenced by the capsular type . Indeed, a 19F serotype strain which was not aggregated by SP-D did exhibit aggregation when the original isogenic strain was capsule switched to capsular serotype 3 . However, although our results show that SP-D is capable of aggregating most pneumococci, no correlation between the degree of aggregation and the capsule or multilocus sequence type of the pneumococcus was clearly apparent . Therefore, although the capsule serotype is not the only determinant of aggregation by SP-D, the data presented here indicate that it does have a role to play.

Biotechniques, 2004 Jan, 36(1), 152 - 4
High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol; Wu S et al.; Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast . We report here that pretreatment of P . pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold . DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect . Cultures grown to 1.15-2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures . A cell concentration of 10(10)/mL gave the highest efficiencies . Digestion of pPIC9K within the AOX1 gene with Sacl gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes . Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 microF, and 186 omega . The transformation efficiency at optimal conditions reached 4 x 10(6) transformants/microgram DNA with pPIC9K . A maximum of 2.6 x 10(5) transformants was produced when 1 microgram of pPIC9K DNA was used.

Acta Biochim Pol, 2003, 50(4), 1111 - 8
The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris; Frajnt M et al.; Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP . Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity . It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 x 10(-6)M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP . It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P.pastoris enzyme . Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels.

J Med Microbiol, 2004 Feb, 53(Pt 2), 119 - 23
Comparison of ITS and IGS1 regions for strain typing of clinical and non-clinical isolates of Pichia anomala; Sutar R et al.; Pichia anomala is an emerging nosocomial pathogen and there is a need for methods that distinguish between different P . anomala strains . In the typing of several clinical as well as non-clinical P . anomala strains, the sequence variation of the internal transcribed spacer (ITS) was found to be inadequate for typing purposes . The intergenic spacer 1 (IGS1) region of the rDNA of several P . anomala strains was therefore investigated in detail . The IGS1 region (which varied from 1213 to 1231 bp in length) was interspersed with repeats and had more variation than the ITS regions . Comparative analysis in cases where analysis by the ITS was ambiguous clearly revealed the IGS1 region to be a more discriminatory tool in the typing of P . anomala strains.

J Biol Chem, 2004 Apr 2, 279(14), 13547 - 54 Epub 2004 Jan 15.
Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities; Hesberg C et al.; Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels . Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A . thaliana possesses two genes in tandem orientation spaced by 704 base pairs . The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms . Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment . Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities . Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves . Because AtXDH1 presumably is the more important isoenzyme in A . thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies . AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa . Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol . AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes . Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

J Invertebr Pathol, 2003 Nov, 84(3), 226 - 33
Microorganisms in the gut of beetles: evidence from molecular cloning; Zhang N et al.; We have regularly cultured yeasts from the gut of certain beetles in our ongoing research . In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results . Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles . Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing . Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches . Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.

Eur J Clin Microbiol Infect Dis, 2004 Feb, 23(2), 127 - 30 Epub 2004 Jan 14.
Infections by the yeast Kodomaea (Pichia) ohmeri: two cases and literature review; Han XY et al.; A 14-year-old boy who was neutropenic following chemotherapy for leukemia developed fungemia caused by the yeast Kodomaea ohmeri ( Pichia ohmeri) . The infection was cured by catheter removal and the use of fluconazole . A 74-year-old man who had undergone surgeries for a subcutaneous tumor developed polymicrobic cellulitis involving Kodomaea ohmeri . Despite surgical debridement and antibiotic therapy, the patient died of complications . Including these 2 cases, there have been 10 Kodomaea ohmeri infections reported thus far, all occurring in patients with pre-existing conditions . There have been seven cases of fungemia and one case each of peritonitis, funguria, and cellulitis . The treatment employed varied depending on the site/source of infection . Seven patients recovered and three died . The microbiological data available suggest that Kodomaea ohmeri can be identified definitively by biochemical tests and is susceptible to amphotericin B and either susceptible to or dose dependently susceptible to itraconazole and fluconazole.

Biotechnol Lett, 2003 Nov, 25(22), 1945 - 8
Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion; Passolunghi S et al.; In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 microg lipase ml(-1) of culture was achieved in sorbitol-based medium . However, a large amount of recombinant protein was retained intracellularly throughout the fermentation, pointing to the transport step as a major bottleneck . Therefore a translational fusion with the green fluorescent protein (GFP) was constructed that was expressed and transported similarly to the native lipase and retained catalytic activity . This analytical tool enables a rapid monitoring of product localization and amount, based on GFP-associated fluorescence.

Biol Chem, 2003 Dec, 384(12), 1553 - 63
Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris . A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme; Bar J et al.; Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides . The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein . However, a soluble form of DP IV is also present in body fluids . Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant . The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney . Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics . However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively) . In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively) . Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.






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