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 i