Pichia

Biotechnol Appl Biochem, 2001 Dec, 34(Pt 3), 199 - 204
Identification of the ligand-binding domain of human vascular-endothelial-growth-factor receptor Flt-1; Ma L et al.; The vascular-endothelial-growth-factor (VEGF) receptor Flt-1 has been shown to be involved in vasculogenesis and angiogenesis . The receptor is characterized by seven immunoglobulin-like loops within the extracellular domain and the first three N-terminal immunoglobulin-like loops are involved in high-affinity binding of VEGF . The minimal extracellular domains of Flt-1 to achieve VEGF binding were screened using the yeast two-hybrid system . The result showed that the binding capacity of loop2-3 was close to that of loop1-3 . The two truncated mutants consisting of loop2-3 and loop1-3 were expressed in the methylotrophic yeast Pichia pastoris at high levels (0.3 mg/litre) . The corresponding proteins, named soluble (s)Flt-1(2-3) and sFlt-1(1-3), were purified . An in vitro biological activity assay showed that the binding capacity of sFlt-1(2-3) to human VEGF(165) and the inhibiting effect of it on human umbilical-vein endothelial cell proliferation stimulated by human VEGF(165) were close to those of sFlt-1(1-3) . Animal tests showed that sFlt-1(2-3) could significantly inhibit the formation of regenerated blood vessels stimulated by hVEGF(165).

FEBS Lett, 2001 Nov 23, 508(3), 332 - 6
Mechanism of Kex2p inhibition by its proregion; Lesage G et al.; Many proteases are produced as zymogens bearing an N-terminal proregion acting both as intramolecular chaperone and as enzyme inhibitor . We studied here the inhibition mechanism of the yeast proprotein convertase Kex2p by its proregion . A recombinant secreted and soluble form of Kex2p was produced in Pichia pastoris and its enzymatic properties toward a fluorogenic synthetic peptide were characterized . Recombinant Escherichia coli-produced Kex2p proregion specifically and potently inhibited the enzyme, with an IC(50) of 160 nM . Exploration of the inhibition mechanism revealed that the proregion behaved as a mixed inhibitor.

Plant Mol Biol, 2001 Nov, 47(5), 607 - 20
Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family; Blee KA et al.; Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean . The most important of these, designated FBPI, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor . Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA . Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst . However, FBP1 when expressed in Pichia pastoris generated H2O2 using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells . FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells . They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots . This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods . Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.

Biochemistry, 2001 Dec 4, 40(48), 14629 - 34
Specificity of the interactions between Glu-3, Ser-24, and Gln-30 within the N-terminal segment of rat liver mitochondrial overt carnitine palmitoyltransferase (L-CPT I) in determining the malonyl-CoA sensitivity of the enzyme; Jackson VN et al.; Using deletion mutants of rat liver-type carnitine palmitoyltransferase I (L-CPT I) expressed in Pichia pastoris, two contiguous discrete sequences within its N-terminal segment have been shown to be positive (residues 3-18) and negative (19-30) determinants, respectively, of the malonyl-CoA sensitivity of the enzyme . The specific interactions among the three individual residues responsible for these opposing effects within these two regions are here investigated in the context of the full-length protein . The pro-inhibitory effects are due to Glu-3 {Shi et al . (1999) J . Biol . Chem . 274, 9421-9426} . We now find that Asp can only partially substitute for Glu-3, whereas the Glu-3Gln mutation has the same effect as the Glu-3Ala mutation . This suggests that a negative charge in this position is essential and that the longer side chain of glutamate is essential for optimal malonyl-CoA sensitivity . Residues within the predicted alpha-helical 19-30 region responsible for decreasing the sensitivity to malonyl-CoA are shown to be neither the three basic (Arg-22, His-25, and Lys-29) nor the two acidic (Asp-20 and Glu-26) residues, as their mutation to Ala produced only small positive effects on malonyl-CoA sensitivity . The residues responsible were identified as Ser-24 and Gln-30, and their effect was shown to be entirely dependent on the presence of Glu-3 . This result reveals that the major sensitization of L-CPT I to malonyl-CoA observed upon deletion of residues 19-30 is not due to a spacer effect with respect to Glu-3 but rather the loss of the two specific residues now identified.

Eur J Biochem, 2001 Nov, 268(22), 5687 - 95
Endoglucanase I from the edible straw mushroom, Volvariella volvacea . Purification, characterization, cloning and expression; Ding SJ et al.; We isolated an endoglucanase, EG1, from culture fluid of Volvariella volvacea grown on crystalline cellulose by ion-exchange and gel filtration chromatography, and preparative PAGE . EG1 has a molecular mass of 42 kDa as determined by SDS/PAGE and an isoelectric point of 7.7 . Enzyme-catalysed hydrolysis of carboxymethyl-cellulose (CM-cellulose) is maximal at pH 7.5 and 55 degrees C . EG1 also hydrolysed phosphoric acid-swollen cellulose and filter paper (at rates of 29% and 6%, respectively, compared with CM-cellulose), but did not hydrolyse crystalline cellulose, cotton, oat spelt xylan, and birchwood xylan . Degenerate primers based on the N-terminal sequences of purified EGI and a protease-generated fragment were used to generate cDNA fragments encoding a portion of the EG1 gene (eg1), and RACE was used to obtain full-length cDNA clones . The cDNA of eg1 contained an ORF of 1167 bp encoding 389 amino acids . The amino-acid sequence from Ala24 to Thr40 corresponded to the N-terminal sequence of the purified protein . The first 23 amino acids are presumed to be a signal peptide . V . volvacea EG1 has been assigned to glycoside hydrolase family 5 according to the classification of glycohydrolases based on amino-acid sequence similarities . Transcripts of eg1 were detected in total RNA from mycelium grown on cellulose but not from mycelium grown on glucose . Cellobiose also induced eg1 expression in 1- to 4-day-old cultures but the signal intensity was lower than that obtained with cellulose . Catabolite repression was observed 24 h after addition of 1% (w/v) glucose, alpha-lactose, beta-lactose, xylose, mannose, sorbose or fructose to medium containing 1% (w/v) crystalline cellulose . Eg1 was expressed at a high level in the yeast, Pichia pastoris, and the catalytic activity of the recombinant EG1 was confirmed.

Protein Expr Purif, 2001 Dec, 23(3), 468 - 75
High-level production and purification of P30P2MSP1(19), an important vaccine antigen for malaria, expressed in the methylotropic yeast Pichia pastoris; Brady CP et al.; P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria . P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria . However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes . Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris . As salt precipitation is a common problem during P . pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation . A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography . P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1 . Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces . P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine .

Protein Expr Purif, 2001 Dec, 23(3), 419 - 25
Production of an anti-prostate-specific antigen single-chain antibody fragment from Pichia pastoris; Wang Y et al.; Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer . Because PSA levels are normally quite low, an antibody-based assay must be used to detect PSA . However, not all PSA-specific antibodies bind equally well to PSA or to its different isoforms . Therefore, a better understanding of how PSA interacts with PSA-specific antibodies is of considerable clinical interest . B80.3 is a widely used murine monoclonal anti-PSA antibody (IgG), which has very high affinity for both free and alpha-anti-chymotrypsin complexed PSA . More importantly, its gene sequence is known-making it one of only two anti-PSA antibodies that has been fully cloned and sequenced . To better elucidate the interaction between PSA and B80.3, a single-chain antibody fragment, derived from the variable domain of B80.3 (scFvB80), was cloned into a pPIC9 vector and expressed in Pichia pastoris . The secreted protein was purified using a three-step protocol beginning with a 50% ammonium sulfate precipitation step, followed by a T-gel thio-affinity step and concluding with a simple anion-exchange (DE52) filtration step . NMR studies indicate the protein is correctly folded while competitive enzyme-linked immunosorbant assays show that the purified scFvB80 has approximately 20% of the activity of the full-length B80.3 antibody . The protocol described here provides a quick and convenient route to prepare large quantities of very pure anti-PSA antibody fragments (15-20 mg/L culture medium) for detailed structural and biophysical characterization .

Bioconjug Chem, 2001 Nov-Dec, 12(6), 1012 - 20
Synthesis of LJP 993, a multivalent conjugate of the N-terminal domain of beta2GPI and suppression of an anti-beta2GPI immune response; Jones DS et al.; LJP 993, a tetravalent conjugate of the amino-terminal domain (domain 1) of beta2GPI, was synthesized, and studies were carried out to explore the ability of LJP 993 to bind anti-beta2GPI antibodies and to function as a B cell toleragen . Domain 1 was expressed in Pichia pastoris, and the N-terminus was site-specifically modified by a transamination reaction converting the N-terminal glycine to a glyoxyl group . A tetravalent platform was synthesized with linkers that terminate in aminooxy groups . This was accomplished by preparing an ethylene glycol-based heterobifunctional linker that contains both a Boc-protected aminooxy group and a free primary amine . The linker was used to modify a tetravalent platform molecule by reacting the amino groups on the linker with 4-nitrophenyl carbonate esters on the platform to provide a linker-modified platform, and the Boc protecting groups were removed to provide a tetravalent aminooxy platform . Glyoxylated domain 1 was attached to the platform to provide LJP 993 by formation of oxime bonds . The protein domains of LJP 993 retain activity as evidenced by the ability of LJP 993 to bind to anti-beta2GPI antibodies . Dissociation constants (Kd) for domain 1 and LJP 993 bound to immobilized affinity-purified anti-beta2GPI antibodies from autoimmune thrombosis patients were determined using surface plasmon resonance . An immunized mouse model was developed to test the ability of LJP 993 to act as a toleragen . A thiol containing domain 1 analogue was expressed in insect cells using the baculovirus expression system, and it was used to prepare an immunogenic conjugate of domain 1 and maleimide-derivatized keyhole limpet hemocyanin (KLH) . Mice were immunized with the KLH conjugate, and spleen cells were harvested from the immunized mice . The cells were incubated with various concentrations of LJP 993 and transferred to mice whose immune systems had been compromised by irradiation . The hosts were then boosted with the KLH-domain 1 conjugate, and after 7 days their antibody levels were measured . Host mice receiving cells that were treated with LJP 993 produced significantly lower amounts of anti-domain 1 antibodies than controls which received untreated cells, indicative of B cell tolerance.

Bioconjug Chem, 2001 Nov-Dec, 12(6), 924 - 31
Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor; Zhou YF et al.; A bifunctional fusion enzyme system constructed by gene splicing is proposed as a new model to develop sequence biosensors, taking maltose biosensor as an example . The cDNA fragment of Aspergillus niger glucoamylase (E.C 3.2.1.3, GA) was fused to the 3' end of Aspergillus niger glucose oxidase (E.C 1.1.3.4, GOD) gene with the insertion of a flexible linker peptide {-(Ser-Gly)5-} coding sequence . The fusion gene was cloned into the vector pPIC9 and expressed in Pichia pastoris GS115 under the control of the AOX1 promoter . It was found that a bifunctional hybrid protein with a molecular weight of 430 kDa was secreted after induction with methanol . The fusion enzyme GOD-(Ser-Gly)5-GA (GLG) was purified using Q Sepharose Fast Flow ion-exchange chromatography . Kinetic analysis demonstrated that GLG retained the typical kinetic properties of both GA and GOD . After being immobilized on an aminosilanized glass slide through covalent bonding by glutaraldehyde, GLG showed much higher sequential catalytic efficiency than the mixture of separately expressed GA and GOD (GA/GOD) . Maltose biosensors were fabricated with GLG and GA/GOD, respectively . The performance characteristics of the maltose biosensor with respect to reproducibility, signal level, and linearity were effectively improved by using the fusion enzyme . Our findings offer a basis for the development of other sequence biosensors.

Protein Eng, 2001 Sep, 14(9), 711 - 5
Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris; Gustavsson M et al.; Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides . The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function . Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed . Three variants were found to be stable throughout 7-day cultivations . The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers . The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wild-type lipase.

Protein Eng, 2001 Sep, 14(9), 705 - 10
Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system; Koganesawa N et al.; Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host . The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals . When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme . On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme . Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal . However, its level of expression was not increased . This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion . Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.

Plant Physiol, 2001 Nov, 127(3), 1180 - 92
Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls; Catala C et al.; The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall . One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules . As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5) . In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin . Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT . LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments . Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment . LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media . Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species . The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Mol Biochem Parasitol, 2001 Nov, 118(1), 61 - 73
Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense; Caffrey CR et al.; Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors . To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris . Rhodesain was secreted as an active, mature protease . Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization . An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma . Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T . b . rhodesiense and all life-cycle stages of T . b . brucei . With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages . Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T . b . brucei . Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain . Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function . S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 474 - 7
{The measurement of intra-cellular AOX in recombinant Pichia pastoris}; Gu XY et al.; The activities of intracellular alcohol oxidase(AOX) in recombinant P . pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment . The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established . The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX . It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0 . Furthermore, the values of qo2max and Km of AOX versus oxygen consumption, which were 0.409 s-1 and 0.16 respectively, were calculated . It is a simple and sensitive and feasible method for quick measuring of AOX.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 456 - 9
{Expression of chimeric protein of Plasmodium falciparum in yeast recombinant Pichia pastoris in cell-high-density fermentation}; Guo MJ et al.; The effects of cultural conditions, which were fermentation period when methanol was as sole carbon source, methanol concentration, range of pH, on the expression of the chimeric protein of Plasmodium falciparum in genetically engineered methylotrophic Pichia pastoris were investigated by shake flask experiments in this paper . The results showed: (1) fermentation period with methanol inducement is about 96 hours; (2) the optimum methanol concentration as sole carbon source is 10 g/L; (3) the range of pH is from 6.0 to 7.0 . On the base of above experimental results the cell high-density fermentation had been done on the FMG-5L fermentor with multi-sensors . The results showed that the cell optical density (OD600) can reached 550 and the maximum expression level of target proteins was 780 mg/L which was 4 times higher or more than the shake flask culture's.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 400 - 5
{Cloning and expression of Aspergillus niger glucose oxidase gene in methylotrophic yeast}; Zhou YF et al.; The DNA fragment encoding A . niger glucose oxidase was amplified by PCR using A . niger genomic DNA as template, and was cloned into vector of pPIC9 for expression in Pichia pastoris . When transformed into methylotrophic yeast Pichia pastoris GS115, The constructed plasmid pPICGOD1 directed the synthesis and secretion of functionally active GOD . After induction in MM medium for 4 days, the GOD activity in the medium reached 30-40 u/mL . SDS-PAGE revealed that recombinant yeast GOD was expressed up to 60%-70% of the total soluble protein, and the secreted GOD could be purified to electrophoretic homogeneity with one purification step using Q Sepharose Fast Flow ion exchange chromatography . The recombinant yeast GOD had very high catalytic activity, showed about 1.6-fold increase of specific activity over the commercial A . niger GOD . Kinetic analysis clearly demonstrated that recombinant yeast GOD showed similar substrate affinity for glucose to A . niger GOD, but the turnover number of the GOD from yeast was determined to be much higher than that of A . niger GOD . In addition, the linear range of glucose electrode made with recombinant yeast GOD was efficiently widened due to the high catalytic activity of yeast GOD.

Arch Biochem Biophys, 2001 Nov 15, 395(2), 199 - 207
cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris; Almeida MS et al.; In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity . Several parameters that affect the level of protein expression were assayed . The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1 . The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region . Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1 . Nevertheless, the rPsd1 presented a more species-specific antifungal activity . The importance of the N- and C-termini for Psd1 activity is pointed out .

J Virol Methods, 2002 Jan, 99(1-2), 99 - 114
Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E . coli and P . pastoris; Watelet B et al.; The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris . A hexahistidine tag was introduced at the C terminus of the E . coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography . The P . pastoris expressed HBcAg was isolated following heat treatment . The two recombinant HBcAgs were purified further on a sucrose gradient . Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P . pastoris and reaction with Ellman's reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E . coli or P . pastoris . Electron microscopy indicated that the E . coli and the P . pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm . Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P . pastoris derived particles suggesting structural discrepancies between the two recombinant molecules . The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum . The preliminary results indicate that the P . pastoris HBcAg produced intracellularly is more suitable than the renatured E . coli HBcAg for detection of anti-HBc in this diagnostic assay.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1703 - 5 Epub 2001 Oct 25.
Expression, characterization and crystallization of the Fv fragment of mouse antibody 3B62 from the secondary immune response; Murase K et al.; Affinity of antibodies increases in the course of the immune response . Mouse anti-nitrophenol antibody 3B62 from the secondary immune response shows higher affinity than the primary-response antibodies . An expression system for the 3B62 Fv fragment was constructed by introducing coding regions for the V(L) and V(H) into the genome of the methylotrophic yeast Pichia pastoris . Each of the coding regions was placed downstream of the coding region for the secretion signal of the yeast alpha-factor . The alpha-factor signals were cleaved off from the expressed proteins and the Fv was secreted as a heterodimer consisting of the V(L) and V(H) domains . The binding constant of the expressed Fv against the (4-hydroxy-5-iodo-3-nitrophenyl)acetate ligand was comparable to that of the Fab fragment . Crystals of the Fv were obtained in the presence of the ligand and diffracted X-rays to 1.8 A resolution . The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 46.48 (9), b = 34.99 (4), c = 77.76 (17) A, beta = 101.47 (14) degrees, and contain one Fv molecule per asymmetric unit.

Biosens Bioelectron, 2001 Dec, 16(9-12), 1001 - 7
Analysis of ethanol-glucose mixtures by two microbial sensors: application of chemometrics and artificial neural networks for data processing; Lobanov AV et al.; Although biosensors based on whole microbial cells have many advantages in terms of convenience, cost and durability, a major limitation of these sensors is often their inability to distinguish between different substrates of interest . This paper demonstrates that it is possible to use sensors entirely based upon whole microbial cells to selectively measure ethanol and glucose in mixtures . Amperometric sensors were constructed using immobilized cells of either Gluconobacter oxydans or Pichia methanolica . The bacterial cells of G . oxydans were sensitive to both substrates, while the yeast cells of P . methanolica oxidized only ethanol . Using chemometric principles of polynomial approximation, data from both of these sensors were processed to provide accurate estimates of glucose and ethanol over a concentration range of 1.0-8.0 mM (coefficients of determination, R(2)=0.99 for ethanol and 0.98 for glucose) . When data were processed using an artificial neural network, glucose and ethanol were accurately estimated over a range of 1.0-10.0 mM (R(2)=0.99 for both substrates) . The described methodology extends the sphere of utility for microbial sensors.

Pflugers Arch, 2001, 442(6 Suppl 1), R184 - 6
Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast pichia pastoris; Lasnik MA et al.; Human granulocyte colony stimulating factor (hG-CSF) was expressed in the methylotrophic yeast Pichia pastoris, using two different constructs which resulted in proteins with different N-terminal sequences . In the first construct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact processing . In the second construct, the gene was fused to the alpha-MF prepro leader at the Lys-Arg processing site (without Glu-Ala spacer) . The PCR products were cloned in pPIC9 commercial vector and integrated into the alcohol oxidase region of the host genome . Transformation was done by electroporation or spheroplasting . Selection of good producing clones was performed by immunoblot analyses of the supernatants from shake-flask fermentation . Proper processing of the products was confirmed by N-terminal sequencing of the secreted proteins . With both plasmid constructs, the target proteins, bearing the histidine tag or not, represented majority of the secreted proteins . Although the proteins were present in the soluble form, they were highly aggregated, which interfered with purification . The most efficient way to obtain monomeric, biologically active protein was complete denaturation by guanidine-HCl or urea and subsequent renaturation during gel filtration chromatography.

Protein Expr Purif, 2001 Nov, 23(2), 301 - 10
Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells; Khandekar SS et al.; CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity . Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154 . However, these studies have not examined the structure or biological function of the carbohydrate on CD154 . Human CD154 contains a single N-linked glycosylation site at asparagine 240 . We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates . Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates . sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells . Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions .

Protein Expr Purif, 2001 Nov, 23(2), 282 - 8
Production of recombinant human bile salt-stimulated lipase in Pichia pastoris; Murasugi A et al.; Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris . Human BSSL has 16 successively repeated sequences in the carboxy terminal region . The sequence consists of 11 amino acid residues . The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression . The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA . Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation . A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days . On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7 . Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium . By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction . The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography .

Biosci Biotechnol Biochem, 2001 Sep, 65(9), 2050 - 7
Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris; Yoshida M et al.; The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris . After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P . chrysosporium . Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting . The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH . The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH . From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.

Biotechnol Bioeng, 2001 Nov 20, 75(4), 485 - 91
Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris; Adrio JL et al.; We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system . Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis . SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS . The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol . Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively .

Plant Physiol, 2001 Oct, 127(2), 674 - 84
Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris; Molhoj M et al.; The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD . As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)) . Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein . A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form . The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD . Delta(1-90)Cel16 was highly N glycosylated as compared with the native B . napus Cel16 protein . Delta(1-90)Cel16 had a pH optimum of 6.0 . The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium . Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose . It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose . Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.

Thromb Haemost, 2001 Sep, 86(3), 902 - 8
A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro; Marques JA et al.; Barbourin is a 73 amino acid venom protein that inhibits platelet aggregation . Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahistidine (H6) tags . BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP . BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin alphaIIbbeta3 . The terminal catabolic half-life of BLAH6 approached that of RSAH6 {3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 +/- SD)}, but was substantially increased relative to that of BARH6 {0.15 +/- 0.03 days (n = 3 +/- SD)} . Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.

J Biochem (Tokyo), 2001 Oct, 130(4), 535 - 42
Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells; Umeda T et al.; Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells . However, few of their substrates are known to have physiological significance in the apoptotic process . We focused our screening for caspase substrates on cytoskeletal proteins . We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat . The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho . These results suggest that filamin is cleaved by a caspase-3-like protease . To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system . When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo . Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo . These results suggest that filamin is an in vivo substrate of caspase-3.

Protein Expr Purif, 2001 Oct, 23(1), 167 - 74
Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris; Briand L et al.; Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species . These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants . In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy . The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide . Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98% . After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography . The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively . The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins .

Protein Expr Purif, 2001 Oct, 23(1), 84 - 96
Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris; Bisht H et al.; The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P . pastoris . The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein . Two in-frame gene fusions were constructed for intracellular expression in P . pastoris . The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E) . In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene . These fusion genes were integrated into the genome of P . pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter . Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P . pastoris based on Northern analysis . The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps . Final purification achieved was approximately 1400-fold with a yield of approximately 26% . The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles) . The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature . On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs . The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein .

Protein Expr Purif, 2001 Oct, 23(1), 55 - 65
Expression and characterization of recombinant human antithrombin III in Pichia pastoris; Mochizuki S et al.; Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade . To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P . pastoris genome, and screened for a single copy transformant . The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h . From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods . We characterized rATIII and compared it with human plasma-derived ATIII (pATIII) . The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII . Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII . O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center . Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half . As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity . pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin .

Curr Opin Investig Drugs, 2001 Apr, 2(4), 477 - 9
Killer anti-idiotypes in the control of fungal infections; Magliani W et al.; Killer anti-idiotypes (KTantild) bear the internal image of a Pichia anomala toxin (KT), characterized by microbicidal activity against prokaryotic and eukaryotic pathogenic microorganisms presenting specific cell wall receptors (KTR) . KTantiId produced by idiotypic vaccination with a KT-neutralizing monoclonal antibody confer active and passive immunoprotection in experimental models of systemic and vaginal candidiasis . KTantild-like human natural anti-KTR antibodies are produced in natural infections caused by KT-sensitive microorganisms . KTantiId in the monoclonal and recombinant forms show therapeutic activity in experimental vaginal candidiasis and Pneumocystis carinii pneumonia . Human commensal bacteria expressing KTantild or killer mimotopes synthesized from the sequence of KtantiId, may represent effective tools to combat fungal infections.

J Protein Chem, 2001 Feb, 20(2), 107 - 13
Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori); Masaki K et al.; Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme . It was expressed by using the methylotrophic yeast Pichia pastoris . The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes . The denaturation temperature was 17-26 degrees C lower than those of human and hen egg-white lysozymes . Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme . It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes . The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes . The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.

Yeast, 2001 Sep 30, 18(13), 1187 - 95
The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames; Ruiz T et al.; We have determined the sequence of a 15 769 bp DNA segment of Pichia anomala . The sequence contains seven complete open reading frames (ORFs) longer than 100 amino acids and a putative tRNA gene . Two of the ORFs code for the well-characterized genes SEC61 (which codes for the core subunit of the ER translocation complex) and FBP1 (encoding fructose-1,6-bisphosphatase) . A gene coding for a protein similar to S . cerevisiae YDL054c was found between the two genes . These three genes show a different organization (intermingled triples) in three yeast species: Saccharomyces cerevisiae, Candida albicans and P . anomala . Two out of the four remaining ORFs show weak homology with different proteins from other species and the other two show non-significant similarity with previously sequenced genes . The nucleotide sequence has been submitted to the EMBL database under Accession No . AJ306295 .

Blood Coagul Fibrinolysis, 2001 Sep, 12(6), 433 - 43
Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris; Sheffield WP et al.; Hirudin is a small, proteinaceous thrombin inhibitor that clears rapidly from the circulation . A hexahistidine-tagged hirudin-rabbit serum albumin (RSA) fusion protein, HLAH6, was characterized following secretion from Pichia pastoris . HLAH6 bound to immobilized nickel, anti-RSA, and anti-hexahistidine antibodies, and contained the expected (ITYTD) N-terminus . Its spectrometric mass was 74,490 (versus the theoretical mass of 74,410 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of 84 kDa) . The terminal catabolic half-life in rabbits of HLAH6, recombinant Pichia-derived His-tagged RSA, or plasma-derived RSA did not differ . Injection of 2 mg/kg HLAH6 into rabbits raised the activated partial thromboplastin time (aPTT) above initial values for 4-24 h, while the equimolar dose of unfused hirudin was without significant effect . A higher dose of HLAH6 (3 mg/kg functional HLAH6, equivalent to 37.6 thrombin-inhibitory units/g) raised the aPTT by 2.0- to 2.5-fold; the elevation persisted for > 48 h . Importantly, both HLAH6 and unfused hirudin inhibited clot-bound thrombin . Our results suggest that HLAH6 exhibits not only delayed clearance, but also prolonged biological activity in vivo compared with unfused hirudin.

Virus Res, 2001 Nov 5, 79(1-2), 125 - 35
Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease; Ruitenberg KM et al.; Equine herpesvirus 1 glycoprotein D (EHV-1 gD) has been shown in mouse models and in the natural host to have potential as a subunit vaccine, using various expression systems that included Escherichia coli, baculovirus and plasmid DNA . With the aim of producing secreted recombinant protein, we have cloned and expressed EHV-1 gD, lacking its native signal sequence and C-terminal transmembrane region, into the methylotrophic yeast Pichia pastoris . The truncated glycoprotein D (gD) gene was placed under the control of the methanol inducible alcohol oxidase 1 promoter and directed for secretion with the Saccharomyces cerevisiae alpha-factor prepro secretion signal . SDS-PAGE and Western blot analysis of culture supernatant fluid 24 h after induction revealed gD-specific protein products between 40 and 200 kDa . After treatment with PNGase F and Endo H, three predominant bands of 34, 45 and 48 kDa were detected, confirming high mannose N-linked glycosylation of Pichia-expressed gD (Pic-gD) . N-terminal sequence analysis of PNGase F-treated affinity-purified protein showed that the native signal cleavage site of gD was being recognised by P . pastoris and the 34 kDa band could be explained by internal proteolytic cleavage effected by a putative Kex2-like protease . Pic-gD, when used in a DNA prime/protein boost inoculation schedule, induced high EHV-1 ELISA and virus neutralizing antibodies and provided protection from challenge infection in BALB/c mice.

Biochem Biophys Res Commun, 2001 Sep 14, 287(1), 122 - 5
Characterization of the HCV core virus-like particles produced in the methylotrophic yeast Pichia pastoris; Acosta-Rivero N et al.; Little is known about the mechanism of hepatitis C virion assembly . So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied . Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures . HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient . This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation . The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm . These results indicated that the HCcAg obtained from P . pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage . Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly .

J Biol Chem, 2001 Nov 9, 276(45), 42422 - 35 Epub 2001 Aug 31.
GSA11 encodes a unique 208-kDa protein required for pexophagy and autophagy in Pichia pastoris; Stromhaug PE et al.; Cells are capable of adapting to changes in their environment by synthesizing needed proteins and degrading superfluous ones . Pichia pastoris synthesizes peroxisomal enzymes to grow in methanol medium . Upon adapting from methanol medium to one containing glucose, this yeast rapidly and selectively degrades peroxisomes by an autophagic process referred to as pexophagy . In this study, we have utilized a novel approach to identify genes required for this degradative pathway . Our approach involves the random integration of a vector containing the Zeocin resistance gene into the yeast genome by restriction enzyme-mediated integration . Cells unable to degrade peroxisomes during glucose adaptation were isolated, and the genes that were disrupted by the insertion of the vector were determined by sequencing . By using this approach, we have identified a number of genes required for glucose-induced selective autophagy of peroxisomes (GSA genes) . We report here the characterization of Gsa11, a unique 208-kDa protein . We found that this protein is required for glucose-induced pexophagy and starvation-induced autophagy . Gsa11 is a cytosolic protein that becomes associated with one or more structures situated near the vacuole during glucose adaptation . The punctate localization of Gsa11 was not observed in gsa10, gsa12, gsa14, and gsa19 mutants . We have previously shown that Gsa9 appears to relocate from a compartment at the vacuole surface to regions between the vacuole and the peroxisomes being sequestered . In the gsa11 mutants, the vacuole only partially surrounded the peroxisomes, but Gsa9 was still distributed around the peroxisome cluster . This suggests that Gsa9 binds to the peroxisomes independent of the vacuole . The data also indicate that Gsa11 is not necessary for Gsa9 to interact with peroxisomes but acts at an intermediate event required for the vacuole to engulf the peroxisomes.

FEMS Microbiol Lett, 2001 Aug 21, 202(2), 227 - 32
Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine; Masih EI et al.; Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine . When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets . In vitro experiments confirm that this yeast can be used as a biological control organism against B . cinerea . An account of the molecular characterisation of P . membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No . AF 270935), as well as the interaction between B . cinerea and the yeast, are given here.

J Biomol NMR, 2001 Jul, 20(3), 251 - 61
Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris; van den Burg HA et al.; Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor . The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L . These protein yields were 24-fold higher in a fermentor than in flask cultures . In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source . In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources . Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase . Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 278 - 82
{Inhibition effect in vitro of purified endostatin expressed in Pichia pastoris}; Feng Y et al.; Endostatin is a newly found inhibitor of angiogenesis, which is identified as c-terminal 184 amino acid fragment of collagen XVIII NC1-domain . A 570 bp cDNA fragment of endostatin has been amplified by PCR from a commercial human fetal liver cDNA library . After subcloned into the yeast vector pPIC9 and subsequence to prove its correctness, Pichia pastoris was transformed with the recombinant pPIC9-endostatin . The expressed endostatin in P . pastoris was purified by heparin-sapherose affinity chromatography . It's purity identified by SDS-PAGE thin layer scanning analysis was up to 98.7% and its Mol . Weight measured by MS was 20.34 kD . The expression level was up to 40 mg/L . The first fifteen amino acid sequence of the N-terminal was completely identical with the inner sequence C-terminal fragment of collagen XVIII NC1 domain as has been designed . Bioassay indicated that the recombinant endostatin can inhibit angiogenesis stimulated by bFGF in CAM test and also the proliferation of both HUVEC and ECV304 in an in vitro test.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 254 - 8
{Overexpression of artificial synthetic gene of Aspergillus niger NRRL3135 phytase in Pichia pastoris}; Bei JL et al.; The phytase gene of Aspergillus niger NRRL3135 was modified with a deletion of intron and signal coding sequence . Then, according to the codon preference of Pichia pastoris, modified phyA gene was artificially synthesized and cloned into expression vector of pPICZ alpha A . The recombinant plasmid was transformed into chromosome of Pichia pastoris X-33 strain by electroporation . The results of SDS-PAGE and enzymatic kinetic analysis proved that the recombinant phytase was secreted into culture medium with nearly same character of natural phytase . After screening for high level productive yeast strains, a strain named SPAN-III produced recombinant phytase with 165,000 u/mL under the condition of shake cultivation . It will satisfy the demand for industrialized production in some degree.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 250 - 3
{Purification and characterization of recombinant human interleukin 11 which expressed by Pichia pastoris}; Huang YS et al.; This study first time report a method to purify the rhIL-11 which expressed by Pichia pastoris . rhIL-11 was secreted into the supernatant and collected by centrifugation . The purity of rhIL-11 reached 97% through the steps of ultrafiltration, SP Sepharose FF, Phenyl Sepharose HP and Sephadex G25 . Analysis of SDS-PAGE, Western-blotting, IEF, RP-HPLC, Mass spectrometer, N and C terminus amino acid sequence and bioactivity was conducted . All the analysis results proved that the rhIL-11 expressed by Pichia pastoris was the same as Neumeg which was expressed in E . coli with fusion expression system . So it is possibly a cheaper and easier method to produce rhIL-11 for clinical use.

Arch Biochem Biophys, 2001 Sep 1, 393(1), 67 - 72
Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains; Lin F et al.; We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1-4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein . With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6x His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol . A mouse DAF CCP1-4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn(187) and Asn(262) to Gln was also produced . Using Ni(2+)-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6x His tags could be one-step isolated to SDS-PAGE purity . Polyclonal antibody against native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the purified product . Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways . With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained . The production of large quantities of functional recombinant mouse DAF CCP1-4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo .

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1575 - 80
Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm; Sugimoto M et al.; An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents . cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship . The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively . The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing . They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region . The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.

Biotechniques, 2001 Aug, 31(2), 306 - 10, 312
Vector for pop-in/pop-out gene replacement in Pichia pastoris; Soderholm J et al.; Gene replacement in yeast is often accomplished by using a counterselectable marker such as URA3 . Although ura3 strains of Pichia pastoris have been generated, these strains are inconvenient to work with because they grow slowly, even in the presence of uracil . To overcome this limitation, we have developed an alternative counterselectable marker that can be used in any P . pastoris strain . This marker is the T-urf13 gene from the mitochondrial genome of male-sterile maize . Previous work showed that expression of a mitochondrially targeted form of T-urf13 in Saccharomyces cerevisiae rendered the cells sensitive to the insecticide methomyl, and similar results have now been obtained with P . pastoris . We have incorporated T-urf13 into a vector that also contains an ARG4 marker for positive selection . The resulting plasmid allows for pop-in/pop-out gene replacement in P . pastoris.

FEBS Lett, 2001 Aug 17, 503(2-3), 173 - 8
Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris; Callewaert N et al.; Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man . A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans . In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase . Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues . Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P . pastoris.

Biol Chem, 2001 Jun, 382(6), 987 - 91
Characterization of C-terminally truncated human tissue inhibitor of metalloproteinases-4 expressed in Pichia pastoris; Stratmann B et al.; The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis . Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover . In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris . The inhibition of MMP-8, -9, -12, -13 and -14 by the N-terminal domain of TIMP-4 was analysed . Using a fluorescent MCA-peptide, Ki values for each subclass of MMPs were determined . With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.

Biotechnol Bioeng, 2001 Sep 20, 74(6), 492 - 7
High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system; Goodrick JC et al.; A continuous fermentation process has been developed in Pichia pastoris (P . pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug . Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter . Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations . Continuous production of the enzyme by P . pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source . The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L . Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight {DCW}) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD) . No proteolytic degradation of the enzyme was seen in the continuous fermentation mode .

J Ind Microbiol Biotechnol, 2001 May, 26(5), 259 - 62
Microbial reduction of alpha-chloroketone to alpha-chlorohydrin; Goswami A et al.; Microbial reduction of alpha-chloroketone to alpha-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide . About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the alpha-chloroketone stereospecifically . Many strains provided the R-alpha-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835 . Few microorganisms showed preferential formation of S-alpha-chlorohydrin after reduction . Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-alpha-chlorohydrin with ee of 88%, 79% and 78%, respectively . The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-alpha-chlorohydrin with > or =95% ee was obtained by appropriate modification of reaction conditions.

Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M, 1999, (93), 181 - 8
Expression of yellow jacket and wasp venom Ag5 allergens in bacteria and in yeast; Monsalve RI et al.; Antigen 5 (Ag5), of unknown biological function, is one of the major venom allergens of vespids and fire ants . We have compared the expression of Ag5 in bacteria and in yeast . Recombinant Ag5 from bacteria formed an insoluble intracellular product, which was not properly folded, but that produced in Pichia pastoris was secreted to the extracellular medium . Immunochemical characterizations showed the secreted Ag5 to have the native structure of the natural protein . This is of interest since the B cell epitopes of Ag5 are mainly of the discontinuous type . These studies were made with Ag5s from yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis), and with hybrid Ag5 molecules that contained partial sequences of these two species . In vitro allergenicity studies with sera from yellow jacket-sensitive patients showed that some of these hybrid molecules had a greatly reduced allergenicity but retained the immunogenicity of the natural allergen . This could be of importance for immunotherapy of this type of allergy.

Hum Mol Genet, 2001 Aug 1, 10(16), 1639 - 48
Enzyme therapy for lysosomal acid lipase deficiency in the mouse; Du H et al.; Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes . Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD) . A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD . The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice . Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots . Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice . Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses) . The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice . Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage . TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine . These studies provide feasibility for LAL enzyme therapy in human WD and CESD.

Biotechnol Prog, 2001 Jul-Aug, 17(4), 629 - 33
Scale-up of a high cell density continuous culture with Pichia pastoris X-33 for the constitutive expression of rh-chitinase; Schilling BM et al.; The feasibility of large-scale production of recombinant human chitinase using a constitutive Pichia pastoris expression system was demonstrated in a 21-L continuous stirred tank reactor . A steady-state recombinant protein concentration of 250 mg/L in the supernatant was sustained for 1 month at a dilution rate of 0.042 h(-1) (equivalent to one volume exchange per day), enabling a volumetric productivity of 144 mg/L d (240 U/L d) . The steady-state dry cell weight concentration in this high cell density culture reached 110 g/L . Considering safety and economical aspects, all large-scale cultivations were conducted without molecular oxygen supplementation . Conventional air sparging was used instead . The oxygen demand of the process was determined by off-gas analysis (OUR = 4.8 g O(2) L(-1) h(-1) with k(L)a = 846 h(-1)) and evaluated with regard to further reactor scale-up.

Biotechnol Appl Biochem, 2001 Aug, 34(Pt 1), 1 - 4
Cloning and expression of the external-glycoprotein gene mutant from HIV-2 in the methylotrophic yeast Pichia pastoris and identification of the glycoprotein; Zhang YJ et al.; To achieve high-level expression of HIV-2(ROD) external glycoprotein gp105 in Pichia pastoris, the gp105 gene mutant tP1, with the 5' non-functional region of the gp105 gene removed, was obtained by PCR amplification and was cloned into secreted expression vector pHILS1 . The His(+)Mut(s) recombinant P . pastoris strain was screened by PCR and induced by methanol . SDS/PAGE and Western-blot analyses showed that mutation of the low-usage codon AGG into synonymous codon CGA and the introduction of the optimal codon TTC made P . pastoris overexpress tP1, an 85 kDa heterologous glycoprotein that was secreted into the medium and recognized specifically by HIV-2 polyclonal antibody . The recombinant strain GS115/tP1 had excellent genetic stability in terms of the properties of growth and expression of gp105, and seven out of 58 recombinant stains with a yield of 29% were selected to be used for further purification of gp105.

Protein Expr Purif, 2001 Aug, 22(3), 436 - 42
Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis; Theis S et al.; The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene . A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification . Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter . The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein . These studies demonstrate that tagged hPEPT1 can be expressed functionally in P . pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine . Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography .

Protein Expr Purif, 2001 Aug, 22(3), 381 - 7
High-yield expression and purification of human interferon alpha-1 in Pichia pastoris; Liu PT et al.; For several years, interferon alpha-1, also known as interferon alpha-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves . Currently, recombinant human interferon alpha-D (rHuIFNalphaD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae . In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNalphaD using the Pichia pastoris system . The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNalphaD with better bioactivity than the commercially available rHuIFNalphaD molecule produced in E . coli .

J Exp Bot, 2001 Aug, 52(361), 1635 - 45
beta-Galactosidases with a lectin-like domain are expressed in strawberry; Trainotti L et al.; Strawberry fruits (Fragaria x ananassa Duch.) undergo a marked softening during their ripening, and the process is accompanied by a release of free sugars with galactose among them . In this work total beta-galactosidase activity was measured in cell wall proteins from strawberry fruits at different developmental stages . Three full-length cDNAs (Fa beta gal1, Fa beta gal2 and Fa beta gal3, respectively) encoding different beta-galactosidases (EC 3.2.1.23) were isolated from a library representing red fruit transcripts . All of them could be detected both in fruits and in vegetative tissues . However, only Fa beta gal1 showed an increasing expression during the ripening stages up to a maximum in the red fruits, while the other two (Fa beta gal2 and Fa beta gal3) were mostly found in green fruits and became barely detectable during ripening proper . The three beta-galactosidase-encoding cDNAs were expressed in the yeast Pichia pastoris, and it was thus possible to demonstrate that each of them encode a beta-galactosidase . The expression of the three beta-galactosidase genes appears to be down-regulated by auxin, as already observed for other ripening-related genes of the non-climacteric strawberry . An unusual characteristic of two strawberry beta-galactosidases (Fa beta gal1 and Fa beta gal2) is that at the C-terminus of the enzymes a domain is found which is structurally related to known animal peptides with a sugar-binding ability.

Protein Eng, 2001 Jun, 14(6), 447 - 54
Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris; Werten MW et al.; A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris . Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation . Despite the fact that gelatins are highly susceptible to proteolysis because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry . Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed . Esterification of the carboxylic amino acid side chains resulted in normal migration . The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry . Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C . The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Dec, 14(4), 309 - 12
{Expression and characterization of human vascular endothelial growth factor in Pichia pastoris}; Ma L et al.; OBJECTIVE: To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expression of recombinant human VEGP165 (hVEGF165) with good biological activity . METHODS: Amplifying hVEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and a secreting signal peptides, the recombinant expression plasmid pPIC9K/VEGF165 was constructed and transformed into KM71 . The multiple insert transformants were screened, fermented in flasks and induced by 1% methanol . RESULTS: After 4 days of methanol induction, the expressed hVEGF165 came up to 30% of total proteins in supernatant by SDS-PAGE . The expressed hVEGF165 was further proved having good antigenicity and high specificity by ELISA and Western blot assay, and having good biological activity to stimulate HUVEC proliferation . CONCLUSIONS: High-level expression of secreted hVEGF165 had been successfully achieved in Pichia pastoris expression system.

Gene, 2001 Jul 11, 272(1-2), 75 - 84
The human cDNA for a homologue of the plant enzyme 1-aminocyclopropane-1-carboxylate synthase encodes a protein lacking that activity; Koch KA et al.; The sequences of genes encoding homologues of 1-aminocyclopropane-1-carboxylate (ACC) synthase, the first enzyme in the two-step biosynthetic pathway of the important plant hormone ethylene, have recently been found in Fugu rubripes and Homo sapiens (Peixoto et al., Gene 246 (2000) 275) . ACC synthase (ACS) catalyzes the formation of ACC from S-adenosyl-L-methionine . ACC is oxidized to ethylene in the second and final step of ethylene biosynthesis . Profound physiological questions would be raised if it could be demonstrated that ACC is formed in animals, because there is no known function for ethylene in these organisms . We describe the cloning of the putative human ACS (PHACS) cDNA that encodes a 501 amino acid protein that exhibits 58% sequence identity to the putative Fugu ACS and approximately 30% sequence identity to plant ACSs . Purified recombinant PHACS, expressed in Pichia pastoris, contains bound pyridoxal-5'-phosphate (PLP), but does not catalyze the synthesis of ACC . PHACS does, however, catalyze the deamination of L-vinylglycine, a known side-reaction of apple ACS . Bioinformatic analysis indicates that PHACS is a member of the alpha-family of PLP-dependent enzymes . Molecular modeling data illustrate that the conservation of residues between PHACS and the plant ACSs is dispersed throughout its structure and that two active site residues that are important for ACS activity in plants are not conserved in PHACS.

J Immunol Methods, 2001 Sep 1, 255(1-2), 103 - 14
High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris; Lange S et al.; In this report, we describe the high-yield secretory expression ( approximately 40 mg x l(-1)) of pure, atrazine-specific Fab fragments (K411B) from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells . Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5-l scale . Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media . Approximately 30% of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions.Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 microg x l(-1), which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in Escherichia coli cells . In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E . coli and to the parental mAb K4E7.

Arch Biochem Biophys, 2001 Aug 1, 392(1), 162 - 7
Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system; Paronetto MP et al.; High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell . The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate . P . pastoris appears to possess a high-affinity iron uptake system similar to that of S . cerevisiae, as far as regulation of expression is concerned . However, the P . pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S . cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading .

J Biol Chem, 2001 Sep 21, 276(38), 35297 - 304 Epub 2001 Jul 20.
The cleavable N-terminal domain of plant endopolygalacturonases from clade B may be involved in a regulated secretion mechanism; Degan FD et al.; Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant . By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain . We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain . We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod . We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod . Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.

Metab Eng, 2001 Jul, 3(3), 236 - 49
Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability; Toivari MH et al.; The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials . Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly . One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway . To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S . cerevisiae that also expresses the P . stipitis genes for XR and XDH . The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels . The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization . In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied . One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon . Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h . In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels . Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain . Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces . The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4) .

Metab Eng, 2001 Jul, 3(3), 226 - 35
Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation; Anderlund M et al.; To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame . Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths . These genes were expressed in Saccharomyces cerevisiae . The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2 . The specific activity of the XDH part of the complexes increased when longer peptide linkers were used . Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers . No degradation products were detected by Western blot analysis . S . cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation . When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S . cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed . The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually . Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol . However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher . The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction .

Mol Genet Genomics, 2001 Jun, 265(4), 604 - 14
Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris; Cosano IC et al.; Mitogen-activated protein kinases (MAPKs) are key enzymes in the signal transduction pathways of eukaryotes . We report the isolation of a Pichia pastoris gene, PIM1, which encodes the first MAPK to be identified in this yeast . Pim1 shows the greatest similarity to fungal MAPKs involved in the maintenance of cell integrity . Disruption of the PIM1 gene results in an osmoremediable thermosensitive phenotype reminiscent of that observed in mutants affected in the MAPK Slt2/ Mpk1 of Saccharomyces cerevisiae, which is involved in ensuring cell wall integrity . Furthermore, pim1 mutants are hypersensitive to caffeine and cell wall-destabilising compounds . Pim1 is phosphorylated at two sites, and thereby activated, in response to heat stress, caffeine and agents that alter the fungal cell wall, which is consistent with a role in adaptation to these conditions . These results support the idea that the MAPK-based mechanisms which regulate cell wall integrity are conserved in yeast species . Pim1 is also doubly phosphorylated in S . cerevisiae in response to stimuli that activate the cell integrity pathway in this yeast . In addition, Pim1 is able to activate the transcription of a reporter gene in one-hybrid experiments, as does its S . cerevisiae counterpart, Slt2 . Interestingly, however, Pim1 does not rescue the mutant phenotype of an slt2delta strain . This indicates some functional divergence in MAPK modulation and signal transmission by cell integrity pathways and provides a tool that may contribute to a better understanding of MAPK signalling.

Glycobiology, 2001 Jul, 11(7), 577 - 86
Synthesis of alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on human tumor cells by recombinant alpha1,3galactosyltransferase produced in Pichia pastoris; Chen ZC et al.; This study describes the processing of human tumor cells or cell membranes to express alpha-gal epitopes (Galalpha1-3Gal-beta1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant alpha1,3galactosyltransferase (ralpha1,3GT), produced in the yeast Pichia pastoris . Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody . This ralpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent . It is effectively produced in P . pastoris under constitutive expression of the P(GAP) promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)(6) tag . This tag enables the simple affinity purification of ralpha1,3GT on a nickel-Sepharose column and elution with imidazole . The purified enzyme appears in SDS-PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme . ralpha1,3GT is very effective in synthesizing alpha-gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound alpha-gal epitopes/min/mg . Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9 )cells) with neuraminidase, ralpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6 )alpha-gal epitopes per cell . Effective synthesis of alpha-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma . These data imply that ralpha1,3GT produced in P . pastoris is suitable for the synthesis of alpha-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.

Rapid Commun Mass Spectrom, 2001, 15(14), 1222 - 8
Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometry; John H et al.; Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity . Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials . This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3) . In contrast, there are conflicting data about the disulfide pattern of endogenous material . This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein . The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode . All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage . For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed .

Biochemistry, 2001 Jul 27, 40(28), 8307 - 16
Functional expression of multidrug resistance protein 1 in Pichia pastoris; Cai J et al.; Overexpression of the multidrug resistance-associated protein (MRP1) causes multidrug resistance in cultured cells . MRP1 transports a large number of glutathione, glucuronide, and sulfate-conjugated organic anions by an ATP-dependent efflux mechanism . Six other MRP proteins exist (MRP2-7), and mutations in some of these genes cause major pathological conditions in humans . A detailed characterization of the structure and mechanism of action of these proteins requires an efficient expression system from which large amounts of active protein can be obtained . We report the expression of a recombinant MRP1 in the methylotrophic yeast Pichia pastoris . The protein is expressed in the membrane fraction of these cells, as a stable and underglycosylated 165 kDa peptide . Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants . MRP1 expressed in P . pastoris binds 8-azido{alpha-(32)P}ATP in a Mg(2+)-dependent and EDTA-sensitive fashion, which can be competed by a molar excess of ADP and ATP . Under hydrolysis conditions (at 37 degrees C), orthovanadate induces trapping of the 8-azido{alpha-(32)P}nucleotide in MRP1, which can be further modulated by known MRP1 ligands . MRP1 is also labeled by a photoactive analogue of rhodamine 123 (IAARh123) in P . pastoris/MRP1 membranes, and this can be competed by known MRP1 ligands . Finally, MRP1-positive membrane vesicles show ATP-dependent uptake of LTC(4) . Thus, MRP1 expressed in P . pastoris is active and shows characteristics of MRP1 expressed in mammalian cells, including drug binding, ligand-modulated formation of the MRP1-MgADP-P(i) intermediate (ATPase activity), and ATP-dependent substrate transport . The successful expression of catalytically active and transport-competent MRP1 in P . pastoris should greatly facilitate the efficient production and isolation of the wild type or inactive mutants of MRP1, or of other MRP proteins for structural and functional characterization.

J Biol Chem, 2001 Sep 7, 276(36), 33621 - 9 Epub 2001 Jul 06.
Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi; Leipelt M et al.; Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria . The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria . In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin . Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides . When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P . pastoris . The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids . The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts . Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.

Enferm Infecc Microbiol Clin, 2001 Jun-Jul, 19(6), 249 - 56
{Antifungal susceptibility of emerging yeast pathogens}; Garcia-Martos P et al.; BACKGROUND: To study the antifungal susceptibility of emerging yeast pathogens to know their possible resistance under the need of applying a treatment . MATERIAL AND METHODS: We investigated the in vitro susceptibility of 69 yeast strains isolates of clinical samples, belonging to 24 different species, to amphotericin B, fluconazole, itraconazole, ketoconazole and 5-fluorocytosine . RESULTS: Only 9 species showed susceptibility to all antifungal agents: Candida famata, C . guillermondii, C . holmii, C . kefyr, C . pelliculosa, C . rugosa, C . utilis, C . zeylanoides y Trichosporon cutaneum; the rest of them presented resistance to some antifungal agent . C . haemulonii, Pichia farinosa and Trichosporon mucoides were resistant to amphotericin B; C . haemulonii, C . inconspicua, C . lusitaniae, C . norvegensis, C . pintolepesii, C . valida, P . ohmeri, Rhodotorula glutinis, R . minuta, R . mucilaginosa and Saccharomyces cerevisiae were resistant to azoles; Blastoschizomyces capitatus and C . lipolytica were resistant to 5-fluorocytosine . CONCLUSIONS: The resistance of emerging yeast pathogens to amphotericin B and 5-fluorocytosine is low, while resistance to azoles is significative, especially to fluconazole (36%) . Many of this yeasts present problems of intrinsic resistance . In yeast infections, the correct identification of species and the study of the in vitro susceptibility is important in order to choose the most adequate antifungal treatment.

J Interferon Cytokine Res, 2001 Jun, 21(6), 361 - 7
Canine interleukin-5: molecular characterization of the gene and expression of biologically active recombinant protein; Yang S et al.; Interleukin-5 (IL-5), which is produced primarily by type 2 T helper lymphocytes (Th2), is an eosinophil differentiation and activation factor . Increased numbers of eosinophils in peripherial blood or tissues (eosinophilia) are observed in asthmatic human patients, in animals with helminth infections, and in dogs with allergic diseases . Antagonism of IL-5 activity is being explored as a potential treatment of a number of disease conditions associated with eosinophils in animal models . In order to study the expression and function of this cytokine in the dog, we have isolated and characterized the canine IL-5 gene . The canine IL-5 polypeptide deduced from the cDNA is composed of 134 amino acids that share varying degrees of homology with IL-5 isolated from several mammals . The genomic structure of the canine IL-5 gene consists of four exons and three introns in the coding region, similar to that of the previously characterized human and mouse IL-5 genes . Recombinant canine IL-5 protein, expressed in Pichia pastoris, is biologically active in a cell proliferation assay . Canine IL-5 gene sequences and the biologically active protein described in this study will be useful reagents for future studies of this cytokine in physiologic processes and in pathologic conditions of the dog.

Protein Expr Purif, 2001 Jul, 22(2), 318 - 24
NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris; de Lamotte F et al.; Postgenomic studies have led to an increasing demand for isotope-labeled proteins . We present a method for producing large quantities of truly native (15)N-labeled protein . Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted . Control of all nitrogen sources permits very high labeling yields . As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification . Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase . The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor . This concentration is reached by most reported preparations in P . pastoris . Further concentration by ultrafiltration would compensate for lower production . This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method .

Protein Expr Purif, 2001 Jul, 22(2), 189 - 99
Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris; Wolff AM et al.; Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems . Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected . In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris . Several growth physiological and genetic approaches for optimization of hexose oxidase production in P . pastoris were investigated . Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation . Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated . Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed . However, we show in this study that HOX is transported out of P . pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter .

J Biol Chem, 2001 Aug 31, 276(35), 32495 - 505 Epub 2001 Jul 02.
Photochemical reaction cycle and proton transfers in Neurospora rhodopsin; Brown LS et al.; It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterologous expression in Pichia pastoris . We report on spectroscopic properties of the Neurospora rhodopsin (NR) . The photocycle was studied with flash photolysis and time-resolved Fourier-transform infrared spectroscopy in the pH range 5-8 . Proton release and uptake during the photocycle were monitored with the pH-sensitive dye, pyranine . Kinetic and spectral analysis revealed six distinct states in the NR photocycle, and we describe their spectral properties and pH-dependent kinetics in the visible and infrared ranges . The phenotypes of the mutant NR proteins, D131E and E142Q, in which the homologues of the key carboxylic acids of the light-driven proton pump bacteriorhodopsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be . This implies that, if the NR photocycle is associated with proton transport, it has a low efficiency, similar to that of haloarchaeal sensory rhodopsin II . Fourier-transform Raman spectroscopy revealed unexpected differences between NR and bacteriorhodopsin in the configuration of the retinal chromophore, which may contribute to the less effective reprotonation switch of NR.

Carbohydr Res, 2001 May 18, 332(2), 183 - 9
Large-scale preparation of the oligosaccharide phosphate fraction of Pichia holstii NRRL Y-2448 phosphomannan for use in the manufacture of PI-88; Ferro V et al.; Mild acid-catalysed hydrolysis of the extracellular phosphomannan of the yeast Pichia holstii NRRL Y-2448 produces a high-molecular-weight phosphomannan core, a low-molecular-weight oligosaccharide phosphate fraction, and a neutral oligosaccharide fraction . A method was developed for the large-scale preparation of the oligosaccharide phosphate fraction, consisting predominantly of the pentasaccharide phosphate, 6-O-PO3H2-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 C 3)-alpha-D-Man-(1 --> 2)-D-Man, for use in the manufacture of the promising new anti-cancer agent, PI-88 . Further insights were also gained into the structure of the phosphomannan by HPLC analysis of the time course of the hydrolysis reaction.

J Biochem (Tokyo), 2001 Jul, 130(1), 19 - 22
An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris; Rodriguez E et al.; We report a new and cost-effective approach to prepare (15)N/(13)C labeled proteins for NMR using the Pichia pastoris expression system . Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-tau) . Our results demonstrate that in order to get full incorporation of (15)N and (13)C, the isotopes are not totally required during the initial growth phase of P . pastoris culture . The addition of small amounts of (15)N and (13)C compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein . Our optimized protocol P4 is two-thirds less costly than the classical method using (15)N and (13)C isotopes during the entire growth phase.

Yeast, 2001 Jun 30, 18(9), 797 - 806
High-level production of human type I collagen in the yeast Pichia pastoris; Nokelainen M et al.; Four human genes, two of them encoding the proalpha1 and proalpha2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris . The proalpha1 and proalpha2 chains expressed formed type I procollagen molecules with the correct 2:1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proalpha chains . Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2:1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen . Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro . The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications .

Acta Biol Hung, 2001, 52(2-3), 265 - 9
Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis; Mergler M et al.; During the aerobic batch cultivation of P . stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner . Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation . This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

Biochemistry (Mosc), 2001 Jun, 66(6), 646 - 57
Isolation, purification, and characterization of catalase from the methylotrophic yeast Pichia pastoris; Potapovich MV et al.; Catalase (CATpp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration . Quantitative parameters of absorption and CD spectra of CATpp solutions and of its membrane-concentrated form (CATpp-conc) were studied . Rates of H2O2 decomposition and kinetic characteristics Km and kcat of CATpp and CATpp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30 degrees C, as well as the effective constant kin of the enzyme inactivation rate during the catalysis and the constant k2 of the interaction rate of the Complex I catalases with H2O2 . Thermal inactivation of CATpp in solutions at 45 degrees C was characterized by the effective rate constant kin*, and the low-frequency (27 kHz) ultrasonic inactivation of CATpp at 20 degrees C was characterized by the first-order rate constant kin(US) . All spectral and kinetic characteristics of CATpp and CATpp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CATcb) . All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CATpp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of kcat/Km, thermal stability comparable with the thermal stability of CAT in terms of kin*, the minimal kin, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm2.

J Ind Microbiol Biotechnol, 2001 Mar, 26(3), 145 - 50
Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis; Nigam JN; Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis . The pretreatments, consisting of boiling and overliming with Ca(OH)2 of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation . Ethanol concentration was increased from 6.7 to 20.2 g l(-1) using adapted P . stipitis (A) and pretreated HSSL . The maximum ethanol yield (Yp/s) and productivity (Qp) were 0.41 g g(-1) and 0.44 g l(-1) h(-1), respectively, at an oxygen transfer rate of 2.0 mmol O2 l(-1) h(-1) . The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor.

Biochim Biophys Acta, 2001 Jul 2, 1527(1-2), 88 - 96
Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana; Wilson IB et al.; The core alpha1,3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic . We report the identification, isolation and characterisation of the cDNAs of three genes (FucTA, FucTB and FucTC) encoding proteins similar to alpha1,3-fucosyltransferases in Arabidopsis thaliana . Reverse transcription-polymerase chain reaction was used to amplify the full length coding sequence of FucTA . The FucTA gene, which consists of seven exons, encodes a presumptive protein of 501 amino acids showing an overall sequence identity of 66% to the protein encoded by the recently isolated mung bean Fuc-T C3 cDNA . FucTA was expressed in Pichia pastoris under the control of the AOX1 gene promoter . The soluble enzyme was found to catalyse the same reaction as mung bean core alpha1,3-fucosyltransferase as judged by analyses of the products by MALDI-TOF and high-performance liquid chromatography . The FucTB cDNA was isolated from a lambda-ZAP library, but the clone used an alternative splicing site between the second and third exon resulting in a premature stop codon . The FucTC gene encodes a protein with less than 40% identity to FucTA across 115 amino acids of a total of 401 amino acids and is a member of a new sub-family of plant alpha1,3/4-fucosyltransferase homologues.

Acta Crystallogr D Biol Crystallogr, 2001 Jul, 57(Pt 7), 1038 - 41 Epub 2001 Jun 21.
Cloning, purification, crystallization and preliminary X-ray diffraction analysis of the antistasin-type inhibitor ghilanten (domain I) from Haementeria ghilianii in complex with porcine beta-trypsin; Rester U et al.; Ghilanten, isolated from the leech Haementeria ghilianii, is a potent two-domain anticoagulant protein homologous to the factor Xa inhibitor antistasin . A synthetic gene encoding the amino-terminal domain of ghilanten (ghilanten-D1) was constructed, expressed in the methylotrophic yeast Pichia pastoris and purified by heparin-Sepharose chromatography . Recombinant ghilanten-D1 inhibits bovine trypsin and human factor Xa with equilibrium inhibition constants (K(i)) of 126 and 1.2 nM, respectively . Ghilanten-D1 has been crystallized in complex with porcine beta-trypsin; three different-looking but isomorphous crystal forms were obtained, each belonging to the orthorhombic space group P2(1)2(1)2(1) . These crystals diffracted to beyond 3.6 A resolution using a rotating-anode X-ray source . A data set complete to 3.7 A resolution was collected.

Biochem Biophys Res Commun, 2001 Jun 22, 284(4), 955 - 60
High-level production of functional muscle alpha-tropomyosin in Pichia pastoris; Hilario E et al.; Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E . coli, the protein needs to be modified at the amino terminus in order to be active . Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin . On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S . E., J . Biol . Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional . In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris . Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase .

Sheng Wu Gong Cheng Xue Bao, 2001 Mar, 17(2), 155 - 60
{Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression}; Cai CQ et al.; Pichia pastoris has become an increasingly popular host for heterologous protein production . However, there is neither a high-efficient transformation method nor a fast colony-PCR assay for the yeast yet . In this paper, we report a transformation procedure by electroporation, which reaches the value of up to 2800 transformants/microgram DNA . By using a cold and heat treatment and a modified PCR buffer, we established a simple and reliable colony-PCR protocol to detect recombinant P . pastoris clones, which is comparable to the conventional assay for E . coli colonies . With these two novel techniques, we have successfully achieved the expression of hirudin, an antithrombin agent, in Pichia pastoris . The secreted hirudin maintains a biological activity of 82 antithrombian units per milliliter supernatant from the media.

Sheng Wu Gong Cheng Xue Bao, 2001 Mar, 17(2), 126 - 30
{Molecular cloning and expression of the cDNA encoding angiogenesis inhibitor K4K5 with Pichia pastoris}; Guan XQ et al.; Kringles of human plasminogen except kringle 4 can inhibit the endothelial cell growth . To determine whether recombinant plasminogen kringle 4-5 (r-K4K5) can inhibit the growth of bovine capillary endothelial (BCE) cell and the angiogenesis of chick embryo chorioallantoic membrane (CAM), we cloned and constructed the modified cDNA region encoding kringles 4 and 5 (K4K5) of human plasmin (ogen) and expressed it with pichia multi-copy expression system . One clone, with the most productive expression was selected from hundreds of transformants . Our data showed that the expression product r-K4K5 (MW 21.5 kD) amounted to 150-250 mg/liter, over 80% of the total secreted protein . It could, dose-dependently, inhibit BCE cell proliferation and inhibit chick embryo CAM angiogenesis.

Biotechnol Bioeng, 2001 Aug 20, 74(4), 344 - 52
Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations; Hellwig S et al.; In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins . Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range . A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent . A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts . In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products . We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris . Specific glycerol feed rates in the range of 38-4.2 mg . g(-1) . h(-1) (mg glycerol per gram fresh weight per hour) were investigated . Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg . g(-1) . h(-1) . At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation . These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research .

Biochim Biophys Acta, 2001 Jun 11, 1547(2), 387 - 96
Purification and characterization of active recombinant rat kallikrein rK9; Zani M et al.; The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate . It has been successfully expressed in the Pichia pastoris yeast expression system . A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA . The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase . Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand . The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies . The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I . Our results confirmed the presence of an extended binding site in the rK9 active site . We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1) . Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

BMC Biotechnol . 2001;1(1):2 . Epub 2001 May 24.
The evaluation of yeast derivatives as adjuvants for the immune response to the Bm86 antigen in cattle; Rodriguez Valle M et al.; BACKGROUND: The Gavac vaccine against the cattle tick Boophilus microplus has proven its efficacy in a large number of controlled and field experiments . However, this vaccine could be further improved by searching for new alternative adjuvants that would induce a stronger long-lasting immune response . We conducted several experiments to assay the adjuvant effect of fractions of the recombinant yeast Pichia pastoris in mouse and cattle models . In previous experiments, the combination of the yeast membrane with saponin was the most effective formulation in stimulating the humoral immune response in mice, eliciting a response higher than Montanide 888 . The response was predominantly of the IgG1 isotype . Here, we evaluated the response in cattle and compared the results with that obtained in mice . RESULTS: Bm86 on the membrane of P . pastoris plus saponin produced high antibody titers in cattle, though the protection level against tick infestations was lower when compared to Gavac, probably due to a decrease in the IgG1/IgG2 ratio . The predictive value of the mouse model was studied through correlation analysis between the isotype levels in mice and the efficacy of formulations in cattle . Good correlation was established between the level of antibodies in mice and cattle, and between the amount of anti -Bm86 IgG1 in mice and the degree of protection in cattle . CONCLUSION: Mouse model have the potential to predict immunogenicity and efficacy of formulations in cattle . These results also support the use of the yeast expression system for recombinant vaccine formulations, enabling the prediction of more cost--effective formulations.

Appl Microbiol Biotechnol, 2001 May, 55(4), 463 - 5
Expression level of heterologous proteins in Pichia pastoris is influenced by flask design; Villatte F et al.; The yeast Pichia pastoris is a convenient production system that enables expression of heterologous proteins in high amounts . As a fermentation method, shaking flasks are very popular because of their simplicity of handling and their low cost . We compared the expression level of the enzyme acetylcholinesterase in a transformed strain of P . pastoris grown in different flasks, presenting various designs but all with the same volume . A several-thousand-fold difference appeared in the expression levels; and the results could not be explained by differences between the flasks in the oxygenation of the medium . The data show that flask design is an important factor to consider for optimising fermentation processes.

Biochim Biophys Acta, 2001 May 28, 1539(1-2), 173 - 80
Identification and characterisation of PEX6 orthologues from plants; Kaplan CP et al.; The sunflower (Helianthus annuus) orthologue of PEX6, an AAA ATPase essential for the biogenesis of peroxisomes in yeasts and mammals, was isolated . HaPex6p is immunologically related to Pichia pastoris Pex6p . Like other genes involved in peroxisome biogenesis and function HaPEX6 mRNA and protein levels peak in early post-germinative growth and mRNA levels also increase in senescent tissue . HaPEX6 identifies probable orthologues in Arabidopsis and rice.

Biotechnol Prog, 2001 May-Jun, 17(3), 503 - 12
Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing . Part 2 . Protein recovery; Thommes J et al.; The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery . Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight . The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system . hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step . The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL) . A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration . For the example of hCTRB isolation from cell containing P . pastoris suspensions, a successful use of this strategy is demonstrated . Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product . Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality . Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density . This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application . The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Biotechnol Prog, 2001 May-Jun, 17(3), 495 - 502
Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing . Part 1 . Fermentation; Curvers S et al.; Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented . Making use of the P . pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm . By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing . This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1) . Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation . This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

J Biol Chem, 2001 Jul 27, 276(30), 28058 - 67 Epub 2001 May 29.
Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster . Potential basis of the neural anti-horseadish peroxidase epitope; Fabini G et al.; For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster . This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity . Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody . Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc . Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila . One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity . This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm) . The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.

Yeast, 2001 Jun, 18(8), 679 - 95
Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress; Sakaki T et al.; The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis . Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism . Sterol glycosides were detected in P . pastoris strain GS115, U . maydis, S . macrospora and R . secalis . This glycolipid occurred in both yeast and filamentous forms of U . maydis but in neither form of C . albicans . This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms . Cerebrosides and sterol glycosides from P . pastoris and R . secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy . The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase . Sterol glycoside from P . pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R . secalis contain two derivatives of ergosterol . The biosynthesis of sterol glucoside in P . pastoris CBS7435 and GS115 depended on the culture conditions . The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations . From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells . This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S . cerevisiae . We suggest P . pastoris and two plant pathogenic fungi to be selected for this approach .

J Biotechnol, 2001 Jun 1, 88(1), 21 - 35
Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter; Vassileva A et al.; High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter . However, the time taken to reach peak product concentration is usually very long ( approximately 240 h) . In this paper, we describe the expression of HBsAg in P . pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter . Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose . Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant . However, this was offset by continuous antigen production by the GAP clone . In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure . The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones . The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg . The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen . These particles were sensitive to thiol reagents . We also explored the possibility of secreting the GAP expressed HBsAg in P . pastoris . In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy . However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association . Our studies show that secretion is not a good strategy for expression of HBsAg in P . pastoris . The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter . Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production . Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.

Biotechnol Bioeng, 2001 Jul 20, 74(2), 164 - 71
Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris; Caubin J et al.; The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines . We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest . We show that C-LYTA can be efficiently expressed and secreted in this host . Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices . It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes .

Arch Biochem Biophys, 2001 Mar 1, 387(1), 93 - 8
Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability; Tang SJ et al.; Although Candida rugosa utilizes a nonuniversal serine codon (CUG) for leucine, it is possible to express lipase genes (LIP) in heterologous systems . After replacing the 19 CUG codons in LIP4 with serine codons by site-directed mutagenesis, a recombinant LIP4 was functionally overexpressed in Pichia pastoris in this study . This recombinant glycosylated lipase was secreted into the culture medium with a high purity of 100 mg/liter in a culture broth . Purified recombinant LIP4 had a molecular mass of 60 kDa, showing a range similar to that of lipase in a commercial preparation . Since LIP4 has only a glycosylation site at position Asn-351, this position may also be the major glycosylation site in C . rugosa lipases . Although the thermal stability of recombinant LIP4 significantly increased from 52 to 58 degrees C after glycosylation, there were no significant differences in the catalytic properties of recombinant glycosylated lipase from P . pastoris and the unglycosylated one from Escherichia coil . These two recombinant LIP4s showed higher esterase activities toward long-chain ester (C16 and C18) and exhibited higher lipase activities toward unsaturated and long-chain lipids . In addition, LIP4 does not show interfacial activation as compared with LIP1 toward lipid substrates of tributyrin and triolein . These observations demonstrated that LIP4 shows distinguished catalytic activities with LIP1 in spite of their high sequence homology.

Arch Biochem Biophys, 2001 Apr 1, 388(1), 171 - 7
Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells; Urbatsch IL et al.; P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy . Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N . Lerner-Marmarosh et al . (1999) J . Biol . Chem . 274, 34711-34718) . The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies . Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn --> Gln in mouse MDR3 Pgp, Asn --> Gln or Ala in human MDR1 Pgp) were expressed in P . pastoris and purified to homogeneity . Yields of mutant Pgp were the same as for parent wild-type proteins . Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins . Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.

Arch Microbiol, 2001 Mar, 175(3), 189 - 97
Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase; Perez JA et al.; The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences . The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage . Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants . One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell . Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits . The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms . The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P . anomala . The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells) . Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P . anomala invertase that are determined by the particular primary structure of its protein moiety.

J Biol Chem, 2001 Jul 20, 276(29), 26995 - 7002 Epub 2001 May 16.
Pre-steady-state kinetic analysis of recombinant Arabidopsis NADH:nitrate reductase: rate-limiting processes in catalysis; Skipper L et al.; Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation . Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase . Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms . Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis . Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present . NADPH reduced FAD at less than one-tenth of NADH rate . Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin . Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover . Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate . However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others . This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.

J Biol Chem, 2001 Jul 20, 276(29), 26980 - 7 Epub 2001 May 16.
Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein; Urbatsch IL et al.; Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form . Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid . The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol . Oxidation with Cu(2+) generated the same higher molecular mass species, primarily at 200 and approximately 300 kDa, in high yield . Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide . Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species . In addition, dimeric P-glycoprotein species ( approximately 300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074 . The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism . Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein.

Biochem Soc Trans, 2001 May, 29(Pt 2), 287 - 92
Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase I; Zammit VA et al.; Elucidation of the membrane topology of carnitine palmitoyltransferase (CPT) I showed that the extreme N-terminus is involved in determining the sensitivity of the liver (L) isoform to malonyl-CoA and suggested that interaction between the two cytosolic segments of the CPT I molecule determines the kinetic characteristics of the enzyme . Work with chimaeric liver/muscle-isoform (L/M) proteins constructed from all six possible combinations of three domains {N-terminus plus transmembrane domain 1 (TM1), loop plus TM2 and C-domain} expressed in Pichia pastoris showed that the precise N-C and TM1-TM2 pairings determine the overall kinetic parameters of the protein . Discrete short sequences within the respective N-terminal regions have negative or positive effects on malonyl-CoA sensitivity (L-isoform) or the K(m) for carnitine (M-isoform) in the full-length proteins, thus imparting to them their distinctive kinetic characteristics . Interactions within N-terminal domains also seem to be important in the targeting of the protein to microsomes in the P . pastoris expression system.

Anal Chem, 2001 May 1, 73(9), 1917 - 26
Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching; Shevchenko A et al.; MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown . The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra . A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions . This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.

Biochemistry, 2001 May 22, 40(20), 5931 - 41
Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg; Guthridge JM et al.; Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains . Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg . Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively . Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a beta-sheet secondary structure with a melting temperature of 59 degrees C . Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2 . Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form . These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand . Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2 . Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric . Its sedimentation coefficient was determined to be 1.36 S . The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed . Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface . The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex . Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.

Biotechnol Bioeng, 2001 Jun 20, 73(6), 433 - 41
Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis; Gaskin DJ et al.; The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate . The recombinant lipase was initially expressed in E . coli . Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined . Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly . The yeast was grown and the supernatants assessed in several assays with long and short chain substrates . Based on this preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity for further analysis . It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters . The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase .

Protein Eng, 2001 Mar, 14(3), 201 - 7
Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris; Kalidas C et al.; Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans . Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG . These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively . The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type . The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2) . Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2) . These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages . The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose . The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions.

Proteins, 2001 Jun 1, 43(4), 499 - 508
Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin; Guo RT et al.; Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma . Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alpha(IIb)beta3 of platelets . Rho produced in Escherichia coli inhibited platelet aggregation with a K(I) value of 263 nM . Although functional, Rho produced in E . coli is misfolded based on our 2D and 3D NMR studies . In order to correct the folding problem, Rho was expressed in Pichia pastoris . The recombinant Rho expressed in P . pastoris inhibited platelet aggregation with a resulting K(I) value of 70 nM . This is the same potency as that of native Rho . CD analysis showed that the secondary structures of Rho are pH-independent and contain 3.5-7.9% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil . The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-edited NMR spectra . These results provide the first direct evidence that highly disulfide-bonded disintegrin can be expressed in P . pastoris with the correct fold . This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants .

Int Arch Allergy Immunol, 2001 Apr, 124(4), 454 - 60
Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity; Takahashi K et al.; BACKGROUND: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens . In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1) . METHODS: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells . Secretion of their proforms was checked by SDS-PAGE or immunoblotting . Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1 . RESULTS: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P . pastoris . Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain . A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P . pastoris . The prosequence of pro-C35S/N53Q was removed by an in vitro activation process . The mature C35S/N53Q showed low protease activity . CONCLUSION: N-glycosylation is essential for secretion in insect SF9 cells but not in P . pastoris . Disulfide bonds are essential for secretion in P . pastoris . A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity . The findings are useful for future design of recombinant products for application in immunotherapy .

Enzyme Microb Technol, 2001 May 7, 28(7-8), 632 - 636
Biochemical approaches to the synthesis of ethyl 5-(s)-hydroxyhexanoate and 5-(s)-hydroxyhexanenitrile; Nanduri VB et al.; Three different biochemical approaches were used for the synthesis of ethyl 5-(S)-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2 . In the first approach, ethyl 5-oxo-hexanoate 3 and 5-oxo-hexanenitrile 4 were reduced by Pichia methanolica (SC 16116) to the corresponding (S)-alcohols, ethyl (S)-5-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2, with an 80-90% yield and >95% enantiomeric excess (e.e) . In the second approach, racemic 5-hydroxyhexanenitrile 5 was resolved by enzymatic succinylation, leading to the formation of (R)-5-hydroxyhexanenitrile hemisuccinate and leaving the desired alcohol 5-(S)-hydroxyhexanenitrile 2 with a yield of 34% (50% maximum yield) and >99% e.e . In the third approach, enzymatic hydrolysis of racemic 5-acetoxy hexanenitrile 6 resulted in the hydrolysis of the R-isomer to provide 5-(R)-hydroxyhexanenitrile, leaving 5-(S)-acetoxyhexanenitrile 7 with a 42% yield (50% maximum yield) and >99% e.e.

Biomol Eng, 2001 Jun, 17(6), 167 - 82
Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug; Patel RN; Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes . The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase . To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine {readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4} with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.) . The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T . intermedius . Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris . Using PDH from E . coli or P . pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5 . The reaction yields of >94% and e.e . of >98% were obtained for allysine ethylene acetal 5 . An enzymatic process was developed for the synthesis of {4S-(4a,7a,10ab)}1-octahydro-5-oxo-4 {{(phenylmethoxy)carbonyl}amino}-7H-pyrido-{2,1-b} {1,3}thiazepine-7-carboxylic acid {BMS-199541-01} . The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-{N{{(phenyl-methoxy)carbonyl} L-homocysteinyl} L-lysine)-1,1-disulphide {BMS-201391-01} to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated . This enzyme was overexpressed in E . coli and a process was developed using the recombinant enzyme.

Ultramicroscopy, 2001 May, 87(4), 165 - 75
Strul--a method for 3D alignment of single-particle projections based on common line correlation in Fourier space; Lindahl M; A central problem of 3D reconstruction in single-particle electron microscopy is the determination of relative orientations of the individual projections contributing to the reconstruction . This article describes an implementation of the method of common lines correlation in Fourier space that allows generation of common lines between an arbitrary number of projections which might posses an arbitrary point group symmetry . Based on this method, it is possible to optimize rotational and translational alignment parameters for individual single-particle projections . The underlying philosophy and details of implementation are discussed, and as an illustration a 3D reconstruction in ice of peroxisomal alcohol oxidase from Pichia pastoris, an octameric assembly with 422-symmetry and a molecular weight of 592 kDa is presented.

Mol Plant Microbe Interact, 2001 May, 14(5), 675 - 7
Characterization of the ToxB gene from Pyrenophora tritici-repentis; Martinez JP et al.; The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota . ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB . Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 563 - 9
Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae; Yasuhara T et al.; cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum . The sequences cover the complete open reading frame encoding the prepro-form . The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues . Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding . The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy . In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding . The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.

Cell Biochem Biophys, 2000, 32 Spring, 51 - 61
Environmental response of yeast peroxisomes . Aspects of organelle assembly and degradation; Sakai Y et al.; Nutritional changes can affect either the assembly or disassembly of yeast peroxisomes . In the past decade, insights regarding the molecular mechanisms of peroxisome assembly have been gained chiefly through the cloning of the PEX genes obtained by complementation of corresponding pex mutants in several yeast strains and Chinese hamster ovary cell lines . Depletion of these peroxins (proteins encoded by PEX genes) by deletion of the corresponding genes affects peroxisomal protein import, biogenesis, or proliferation . To complement these studies in the field, the authors undertook an investigation of the functions of a subset of Candida boidinii peroxisomal membrane proteins (PMPs), Pex11, Pmp47, and Pmp20, by analyzing strains of C . boidinii in which the genes encoding these proteins were deleted . The authors' studies show that Pex11p is involved in peroxisome proliferation; Pmp47 plays a role in the translocation, folding, or assembly of dihydroxyacetone synthase; and Pmp20 is probably involved in methanol metabolism . In contrast to the studies on peroxisome assembly, the molecular mechanisms of peroxisome degradation remain poorly understood . To shed light on this problem, the authors isolated Pichia pastoris mutants defective in peroxisome autophagy (pag mutants) . A novel, double-fluorescence method used for the characterization of wild-type cells and of pag mutants enabled us to dissect the microautophagic degradation of peroxisomes into several distinct stages . These studies show that specific PAG gene products are involved in multiple steps of the process . Future cloning and characterization of the functions of PAG genes will reveal the molecular basis of peroxisome degradation.

Biochemistry, 2001 Feb 20, 40(7), 2260 - 6
Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes; Nicot C et al.; The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids . The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition . Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI . When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI . However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes . Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.

Yeast, 2001 May, 18(7), 643 - 55
Efficient expression, purification and characterization of mouse salivary alpha-amylase secreted from methylotrophic yeast, Pichia pastoris; Kato S et al.; We constructed a secretion vector of mouse salivary alpha-amylase, pPAM, using the AOX1 promoter-terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris . Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (approximately 240 microg/ml) of mouse alpha-amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90-fold higher than that of Saccharomyces cerevisiae . Growth temperature of cells was critical for efficient production of alpha-amylase . P . pastoris transformants secreted both core-glycosylated and non-glycosylated alpha-amylase molecules with a glycosylated:non-glycosylated ratio of about 20:80 . Both glycosylated and non-glycosylated alpha-amylases were purified separately to apparent homogeneity . The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non-glycosylated alpha-amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry . We further studied the outer chain glycosylation of engineered mouse alpha-amylase secreted by P . pastoris .

Yeast, 2001 May, 18(7), 621 - 41
Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins; Johnson MA et al.; The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes . Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans . Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris . Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membrane and binds peroxisomal targeting signal (PTS) receptors carrying proteins bound for the peroxisomal matrix, results that have led to the hypothesis that Pex14p is a receptor-docking protein . P . pastoris Pex14p (PpPex14p) behaves like an integral membrane protein, with its C-terminus exposed on the cytosolic side of the peroxisomal membrane . PpPex14p complexes with many peroxins, including Pex3p (Snyder et al., 1999b), Pex5p, Pex7p, Pex13p, Pex17p, itself, and a previously unreported peroxin, Pex8p . A portion of Pex14p is phosphorylated, but both phosphorylated and unphosphorylated forms of Pex14p interact with several peroxins . The interactions between Pex14p and other peroxins provide clues regarding the function of Pex14p in peroxisomal protein import .

J Drug Target, 2000, 8(6), 403 - 12
Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor; Boado RJ et al.; The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris . Restriction endonuclease sites located at both 5'- and 3'-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e . EcoRI and SmaI or AvrII) . The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1 . An OX26 scFv high producer clone {GS115 His+ Mut+ (pPIC-OX26 SacI)} was isolated and used for large-scale production and characterization . Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography . The identity of the OX26 scFv was confirmed by Western blot analyses with both anti c-myc and anti poly-His antibodies . Minor immunoreactive bands corresponding to hyperglycosylated and partially processed alpha-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7 . The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.

J Clin Microbiol, 2001 May, 39(5), 1702 - 6
Outbreak of Pichia anomala infection in the pediatric service of a tertiary-care center in Northern India; Chakrabarti A et al.; An outbreak of nosocomial fungemia due to the unusual yeast, Pichia anomala occurred in the pediatric wards of our hospital over a period of 23 months (April 1996 to February 1998) . A total of 379 neonates and children (4.2% admissions) were infected . The probable index case was admitted to the pediatric emergency ward, with subsequent transmission to the premature nursery, pediatric intensive care units, and other children wards . Carriage on the hands of health care personnel was likely to be responsible for dissemination of the fungus . The outbreak could only be controlled after a health education campaign to improve hand-washing practices was instituted and after nystatin-fluconazole prophylaxis to all premature neonates and high-risk infants was introduced . In a case-control study, we identified a lower gestational age, a very low birth weight (<1,500 g), and a longer duration of hospital stay as significant risk factors associated with P . anomala fungemia in premature neonates . We conducted a culture prevalence survey of 50 consecutive premature neonates and found that 28% were colonized with P . anomala at a skin or mucosal site on the date of delivery and that 20% of these neonates subsequently developed P . anomala fungemia . We performed multilocus enzyme electrophoresis on 40 P . anomala outbreak isolates (including patient and health care workers' hand isolates), and the results suggested that these isolates were identical . Our study highlights the importance of P . anomala as an emerging nosocomial fungal pathogen.

J Biol Chem, 2001 Jul 27, 276(30), 28268 - 73 Epub 2001 Apr 26.
Placental protein 14 induces apoptosis in T cells but not in monocytes; Mukhopadhyay D et al.; Substantial evidence exists in literature to suggest that placental protein 14 (PP14) (recently renamed glycodelin A), exhibits immunosuppressive properties and is an indispensable macromolecule in the maternal system for the establishment, maintenance, and progression of pregnancy . Though there are several reports substantiating the above, the mechanism of its action at the molecular level has not been elucidated as yet . In this paper we provide data that suggest that amniotic fluid PP14 and recombinant PP14 expressed in Pichia pastoris induce apoptosis in human peripheral blood lymphocytes upon activation, independent of monocytes . That PP14 has a direct apoptotic action on T cells but not on monocytes was also demonstrated by utilizing human cell lines . PP14 was shown to induce apoptosis in the human T cell lines, Jurkat and MOLT-4 cells, but not in the human monocytic cell line, U937.

Virus Genes, 2001 Mar, 22(2), 167 - 73
Molecular cloning and high-level expression of G2 protein of hantaan (HTN) virus 76-118 strain in the yeast Pichia pastoris KM71; Ha SH et al.; Hantaan viral G2 envelope gene, which is known to be one of major antigens and induce neutralizing antibodies, was cloned into expression vector pHIL-S1 which consists of AOX1 promoter, PHO1 signal sequence, HIS4 gene and other components . The recombined plasmid was transformed into methylotropic yeast, Pichia pastoris of KM71 and recombinant strains harboring multi-copy of G2 gene were selected . Expression of the cloned G2 gene was confirmed with Western blot analysis using anti-sera of guinea pig immunized with the carboxyl terminal region of G2 protein expressed in Escherichia coli . The expression of G2 gene from the recombinant strain was tightly repressed by dextrose and effectively induced by methanol, an inducer of AOX1 promoter . The highest expression level was observed from 1 day after induction and maintained at the same level for up to 4 days.

Bioseparation, 2000, 9(4), 223 - 30
Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption; Trinh L et al.; Endostatin, a 20 KDa fragment of collagen XVIII, was shown to have an inhibitory effect on angiogenesis and can potentially be used as a tumor growth suppressor . To obtain the amount needed for testing, the protein was successfully cloned and expressed in Pichia pastoris . At the end of the fermentation process, the concentration of the endostatin in the culture was 50 mg per liter, accompanied by 400 gr per liter (wet weight) of biomass . Before the protein can be captured and purified on a packed bed of heparin-Sepharose, the biomass must be removed . Because of the high biomass concentration, conventional biomass removal techniques like centrifugation or filtration are inefficient and cumbersome . Therefore, the expanded-bed adsorption technique was chosen as an alternative approach . An efficient procedure for the initial recovery and purification of the endostatin was developed . The process utilized a cation- exchanger resin instead of a heparin-based affinity resin, because its dynamic capacity was higher, even though it was affected by the high linear flow on the expanded bed . After adjusting the conductivity, pH and biomass concentration, the complete broth was pumped directly on the expanded-bed matrix (Streamline SP XL) . Though the yields of protein are similar, the expanded-bed approach is superior to the packed-bed method for several reasons . The expanded-bed process was shorter (only 8 hours compared to 16 hours for the packed bed), it is cheaper, and the product has higher specific activity (29% compared with 18%) . Endostatin produced by the expanded-bed adsorption method showed the expected bioactivity and is currently being tested for its potential as a tumor suppressor.

Appl Environ Microbiol, 2001 May, 67(5), 2298 - 303
The cel4 gene of Agaricus bisporus encodes a beta-mannanase; Tang CM et al.; Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes . We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris . CEL4 had no detectable activity on cellulose or xylan . This gene is the first isolated from this economically important fungus to encode a mannanase . P . pastoris secreted about three times more CEL4 than S . cerevisiae . The removal of the cellulose-binding domain of CEL4 lowered the secreted specific activity by P . pastoris by approximately 97% . The genomic sequence of cel4 was isolated by screening a cosmid library of A . bisporus C54-carb8 . The open reading frame was interrupted by 12 introns . The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A . bisporus fruiting cultures . In laboratory liquid cultures of A . bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days . The levels of CEL4 broadly mirrored the levels of enzyme activity . In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined . Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation . The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.

J Cell Biol, 2001 Apr 16, 153(2), 381 - 96
Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole; Kim J et al.; Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae . The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation . Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo . In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling . Most of the import machinery characterized thus far is required for all three modes of transport . However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9 . In S . cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy . In P . pastoris, Gsa9 is required for glucose-induced pexophagy . Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy . The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation . Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy . In P . pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy . These biochemical and morphological results demonstrate that Cvt9 and the P . pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Parasite Immunol, 2001 May, 23(5), 237 - 49
Vaccination with neutrophil inhibitory factor reduces the fecundity of the hookworm Ancylostoma ceylanicum; Ali F et al.; Neutrophil inhibitory factor (NIF), a protein isolated from hookworms of the genus Ancylostoma, inhibits CD11b/18-dependent leucocyte function, binding to the I domain of CD11b . Historically, NIF was serendipitously isolated from whole worm extracts during a search for novel antihaemostatic agents, and little is known of its source or biological significance to the parasite . NIF has also been identified as a possible hookworm vaccine candidate . Ancylostoma ceylanicum recombinant NIF, expressed in its active form in Pichia pastoris, was purified and its functional activity confirmed using neutrophil adhesion assays and confirmatory immunoassay . Recombinant NIF was subsequently used in vaccination trials in the A . ceylanicum-hamster model system for human hookworm infection . Vaccinated and challenged animals were not protected in terms of worm burden or haematocrit values, despite the presence of high levels of specific antibody against NIF . However, adult worms resident in vaccinated animals showed a significant reduction in fecundity (85.8% by day 21 postinfection), indicating a degree of protection against subsequent transmission by vaccination . These data indicate that targeted vaccination with recombinant subunit material, derived from a known and effective immune suppressant secreted by the parasite, may offer partial protection against the transmission of hookworm infection . Furthermore, we can also report that a biological activity characteristic of NIF is detectable in the secretions of A . ceylanicum using two complementary bioassays . Complete neutralization of this secreted activity by vaccination in combination with other vaccine candidates may result in improved protection against A . ceylanicum infection.

J Appl Microbiol, 2001 Apr, 90(4), 668 - 75
Characterization of polar lipids of oral isolates of Candida, Pichia and Saccharomyces by Fast Atom Bombardment Mass Spectrometry (FAB MS); Mahmoudabadi AZ et al.; AIMS: To characterize fatty acid and phospholipid analogue profiles of oral yeasts . METHODS AND RESULTS: Twenty-seven strains of oral yeasts were cultured on SDA and lipids of freeze-dried cells were extracted and analysed by FAB MS . The most abundant carboxylate anion was m/z 281 (C18 : 1) . The most intense phospholipid analogue ions were of PE, PG, PA and PI . Pichia etchellsii contained molecular species of PG and PE, whereas Saccharomyces cerevisiae had PA, PG and PE analogues . Mass spectra revealed that S . cerevisiae and Candida glabrata were distinct from one another and from the other species tested . CONCLUSION: Oral yeasts largely differ with respect to their polar lipids . It is concluded that oral yeast species have distinctive fatty acid and phospholipid analogue anion profiles . SIGNIFICANCE AND IMPACT OF THE STUDY: FAB MS provided novel chemotaxonomic information.

J Agric Food Chem, 2001 Apr, 49(4), 1761 - 6
Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris; Choi BK et al.; Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta-casein expressed in the methylotrophic yeast Pichia pastoris . Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation . Glycosylated bovine beta-casein (L70S/P71S) expressed in P . pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A--Sepharose affinity column, and Mono Q anion-exchange FPLC . The purified glycosylated bovine beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type . Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P . pastoris, and its molecular weight was estimated to be 33.6 kDa . Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.

Toxicon, 2001 Aug, 39(8), 1211 - 8
Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris; Borgheresi RA et al.; Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle . They are structurally and functionally related to endothelins . The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer . In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium . Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred . Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium . Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively . The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown . Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process . Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 805 - 14
Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1; Hult M et al.; The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones . Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens . The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris . In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound . Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained . In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 749 - 59
Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?
Blum A, Raum A, Martin H, Maser E.
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification . Since 11beta-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state . Upon expression experiments in Escherichia coli, 11beta-HSD 1 turns out to be hardly soluble without detergents . This study describes attempts to increase the solubility of 11beta-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E . coli and the yeast Pichia pastoris . Furthermore, we investigated if the codon for methionine 31 in human 11beta-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment . Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme . In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor . However, activity could not be found, neither with the truncated protein expressed in E . coli nor with that expressed in P . pastoris . Hydrophobicity plots proved the hydrophobic nature of 11beta-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.

J Chromatogr B Biomed Sci Appl, 2001 Mar 25, 753(1), 51 - 65
Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation; Rolland D et al.; Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up . It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle . The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy . The expression and purification of the different forms of recombinant HBc have been described . For the last step, ultracentrifugation and size-exclusion chromatography were compared . The morphology of these capsids was observed using an electron microscope . Our data shows that HBc antigen is produced in large quantities in E . coli but some contaminants remained which were associated with the E . coli HBc protein after ultracentrifugation or size-exclusion chromatography . The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate . P . pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

J Appl Microbiol, 2001 Mar, 90(3), 397 - 406
Optimization of the wheat puroindoline-a production in Pichia pastoris; Issaly N et al.; AIMS: A recombinant puroindoline-a (rPIN-a) was produced using the methylotrophic yeast Pichia pastoris . METHODS AND RESULTS: In fed-batch culture, the production of rPIN-a decreased after 24 h of methanol induction . Most of the rPIN-a was not soluble in the culture medium remaining bound to the cell walls . Soluble and membrane-bound rPIN-a were quantified by ELISA after Triton X-114 phase partitioning . In order to improve the production of rPIN-a, the influence of pH, specific growth rate and the addition of TX-114 was tested on two independent continuous cultures . The production of rPIN-a was improved when continuous culture was carried out at 29 degrees C under acid conditions (pH 5) with a low dilution rate (D=0.025 h(-1)) . The addition of 0.01% TX-114 to the medium inverted the ratio between the secreted and the membrane-bound rPIN-a . CONCLUSION: When a continuous culture was carried out under optimized conditions, the rPIN-a production yield was increased 10-fold to 14 mg l(-1) and 80% of the rPIN-a was soluble . SIGNIFICANCE AND IMPACT OF THE STUDY: This study would be helpful to optimize the expression of other membrane-bound proteins in P . pastoris.

J Immunol Methods, 2001 May 1, 251(1-2), 123 - 35
Expression of single-chain Fv-Fc fusions in Pichia pastoris; Powers DB et al.; Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments . For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life . We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris . The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer . The increased size of the dimer (approximately 106 kDa vs . approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice . The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors . Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.

Hybridoma, 2001 Feb, 20(1), 35 - 41
Development of a monoclonal antibody specific to a recombinant envelope protein from dengue virus type 4 expressed in Pichia pastoris; Pupo-Antunez M et al.; A mouse monoclonal antibody (MAb, 4B6) was able to recognize dengue virus type 4 envelope (E) protein both as a recombinant protein in Pichia pastoris and when it was present in infected brains of suckling mice . 4B6 was characterized by enzyme-linked immunoadsorbent assay (ELISA), hemaglutination inhibition, neutralization, and immunoblot . The MAb was isotyped as IgG2a . It was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus . It did not enhance infection of P338D1 cells by dengue type 4 virus strain H-241 strain . This MAb was reactive with recombinant E protein and dengue 4 virus, as revealed by Western blot . In vivo, MAb 4B6 conferred passive protection in mice challenged with homologous virus . Currently, this MAb is being used to purify recombinant E protein for further studies.

Biochemistry, 2001 Apr 10, 40(14), 4454 - 8
A novel inhibitor of the mammalian peptide transporter PEPT1; Knutter I et al.; This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter . We provide evidence that the dipeptide derivative Lys{Z(NO(2))}-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM . Characterization of the interaction of Lys{Z(NO(2))}-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1 . By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys{Z(NO(2))}-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys{Z(NO(2))}-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself . Lack of transport was substantiated by the absence of Lys{Z(NO(2))}-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor . The fact that Lys{Z(NO(2))}-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions . Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.

Protein Expr Purif, 2001 Apr, 21(3), 438 - 45
Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris; Li Z et al.; Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth . Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity . In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H . Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein . Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature . These data suggested that P . pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies .

Protein Expr Purif, 2001 Apr, 21(3), 386 - 92
Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain; Rotticci-Mulder JC et al.; Candida antarctica lipase B (CALB) and C . antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris . The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum . The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter . The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L . The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69% . Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated . The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source . The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB .

J Biol Chem, 2001 May 25, 276(21), 18485 - 90 Epub 2001 Feb 21.
Localization of disulfide bonds in the frizzled module of Ror1 receptor tyrosine kinase; Roszmusz E et al.; The frizzled (FRZ) module is a novel module type that was first identified in G-protein-coupled receptors of the frizzled and smoothened families and has since been shown to be present in several secreted frizzled-related proteins, in some modular proteases, in collagen XVIII, and in various receptor tyrosine kinases of the Ror family . The FRZ modules constitute the extracellular ligand-binding region of frizzled receptors and are known to mediate signals of WNT family members through these receptors . With an eye toward defining the structure of this important module family, we have expressed the FRZ domain of rat Ror1 receptor tyrosine kinase in Pichia pastoris . By proteolytic digestion and amino acid sequencing the disulfide bonds were found to connect the 10 conserved cysteines in a 1-5, 2-4, 3-8, 6-10, and 7-9 pattern . Circular dichroism and differential scanning calorimetry studies on the recombinant protein indicate that the disulfide-bonded FRZ module corresponds to a single, compact, and remarkably stable folding domain possessing both alpha-helices and beta-strands.

J Biol Chem, 2001 May 4, 276(18), 15240 - 8 Epub 2001 Feb 07.
Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin; Maeshima Y et al.; Components of vascular basement membrane are involved in regulating angiogenesis . Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity . In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner . The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells . Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest . Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice . The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature . These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin . In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis . Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.

Eur J Biochem, 2001 Apr, 268(7), 1918 - 28
The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases; Tschesche H et al.; The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9) . Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases . We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly . A significant enhancement of the activity could be demonstrated for the HgCl2- and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8 . The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli . This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL . Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL . In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.

J Biotechnol, 2001 Apr 27, 87(1), 17 - 27
Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis; Nigam JN; Ethanol production was evaluated from wheat straw (WS) hemicellulose acid hydrolysate using an adapted and parent strain of Pichia stipitis . NRRL Y-7124 . The treatment by boiling and overliming with Ca(OH)(2) significantly improved the fermentability of the hydrolysate . Ethanol yield (Yp/s) and productivity (Qp av) were increased 2.4+/-0.10 and 5.7+/-0.24 folds, respectively, compared to neutralized hydrolysate . Adaptation of the yeast to the hydrolysate resulted further improvement in yield and productivity . The maximum yield was 0.41+/-0.01 g(p) g(s)(-1), equivalent to 80.4+/-0.55% theoretical conversion efficiency . Acetic acid, furfurals and lignins present in the hydrolysate were inhibitory to microbial growth and ethanol production . The addition of these inhibitory components individually or in various combinations at a concentrations similar to that found in hydrolysate to simulated medium resulted a reduction in ethanol yield (Yp/s) and productivity (Qp av) . The hydrolysate used had the following composition (expressed in g x l(-1)): xylose 12.8+/-0.25; glucose 1.7+/-0.3; arabinose 2.6+/-0.21 and acetic acid 2.7+/-0.33.

J Agric Food Chem, 2001 Feb, 49(2), 641 - 6
High-level production of recombinant chicken cystatin by Pichia pastoris and its application in mackerel surimi; Chen GH et al.; A high level of the secreted form of recombinant chicken cystatin was expressed in Pichia pastoris X-33 by chromosomal integration of multiple copies of an expression cassette containing chicken cystatin under the control of glyceraldehyde-3-phosphate dehydrogenase promoter . The inhibition ability of the recombinant for papain-like proteinase was found to correspond to those of natural chicken cystatin . The recombinant cystatin substantially inhibited the proteolysis of myosin and gel softening, which consequently improved the gel properties of mackerel surimi.

Biotechnol Bioeng, 2001 Apr 5, 73(1), 74 - 9
Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product; Wang Y et al.; The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor . A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method . In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L . A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80% . After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained . In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form .

Histochem J, 2000 Dec, 32(12), 711 - 6
Characterization of monoclonal antibodies to glycodelin and recombinant glycodelin; Karri S et al.; Glycodelin-A belongs to the lipocalin superfamily . Although it is associated with normal endometrial growth during the menstrual cycle, fertilization and normal pregnancy in humans, the molecular mechanism of its biological action has not been elucidated . To undertake studies to understand the functional relevance of any molecule, obtaining large quantities of the protein becomes essential . With the ultimate aim of purifying glycodelin either from its natural sources (human amniotic fluid) or the recombinant glycodelin from bacterial recombinant lysates, we raised monoclonal antibodies to this protein . As immunogens, recombinant glycodelin expressed in E . coli and Pichia pastoris as well as glycodelin from amniotic fluid were used . The monoclonal antibodies generated were characterized with respect to binding to both the native as well as the recombinant proteins using ELISA, immunoblotting, and immunohistochemistry.

Mol Biotechnol, 2000 Nov, 16(3), 193 - 202
Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression . Application to a rabbit liver carboxylesterase; Morton CL et al.; Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells . Although, recombinant protein was observed in E . coli sonicates, little or no enzymatic activity was detected . Similarly, no activity was observed following expression in S . cerevisiae . In contrast, active protein was produced in P . pastoris, from S . frugiperda following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA . For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P . pastoris has proved sufficient . However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S . frugiperda was the most efficient . Using this system, we have generated and purified milligram quantities of essentially pure protein . These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.

Clin Exp Allergy, 2001 Feb, 31(2), 313 - 21
Immunological and molecular characterization of the major allergens from lilac and privet pollens overproduced in Pichia pastoris; Gonzalez E et al.; The main allergens from privet and lilac pollens, Lig v 1 and Syr v 1, are proteins homologous to Ole e 1 and have been shown to be involved in cross-reactivity . To overproduce the correctly folded Lig v 1 and Syr v 1 allergens and to study their immunological properties in comparison with those of their natural counterparts . The yeast Pichia pastoris was used as an expression system to produce these recombinant allergens . The proteins were isolated by ion-exchange and size-exclusion chromatographies . Amino acid quantifying, Edman degradation, mass spectrometry and circular dichroism were carried out to obtain molecular properties of the recombinant proteins . Anti-Ole e 1 monoclonal and polyclonal antibodies, as well as sera from patients allergic to olive pollen, were used in immunoblotting and ELISA for immunological characterization . Recombinant Lig v 1 and Syr v 1 were secreted at high yield to the extracellular medium of the yeast . The purified proteins displayed the native conformation, as deduced from their spectroscopic properties and binding ability to an IgG monoclonal antibody . The recombinant allergens behaved similarly to their natural counterparts when they were analysed against Ole e 1-specific antibodies . IgE and IgG binding properties of lilac and privet allergens to olive allergic sera and Ole e 1-specific antibodies indicated that these molecules share common B-cell epitopes with Ole e 1 . P . pastoris yeast is an appropriate system for the efficient production of Ole e 1-like allergens, which could be used as analogous allergens and predictors of clinical sensitization.

Pancreas, 2001 Mar, 22(2), 186 - 92
Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates; Schiesser M et al.; Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly up-regulated early in acute pancreatitis . PAP expression returns to undetectable levels when the pancreas recovers . In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis . Their functions remain obscure . Pancreatic stone protein (PSP/reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions . PSP/reg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond . Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP) . PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro . PTP can be found in protein plugs or stones from patients with chronic pancreatitis . Rat PAP contains a trypsin cleavage site at the same position as PSP/reg . We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH . To test our hypothesis, we generated and purified recombinant PAP . Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris . We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP) . PATP displays pH-dependent solubility characteristics very similar to those of PTP.

Dev Comp Immunol, 2001 May, 25(4), 345 - 52
Soluble type-I interleukin-1 receptor blocks chicken IL-1 activity; Klasing KC et al.; The ligand-binding domain of the chicken type-I interleukin-1 (IL-1) receptor (soluble IL-1R(I); sIL-1R(I)) was cloned into a Pichia pastoris expression system and the resulting sIL-1R(I) binding protein was used to produce antisera in rabbits (anti-IL-1R(I)) . Two experiments were conducted to determine the capacity of sIL-1R(I) or anti-IL-1R(I) to block the IL-1 bioactivity (thymocyte co-stimulation) in conditioned media (CM) from HD11 chicken macrophages stimulated with lipopolysaccharide . In the first experiment, pre-incubation of CM with unpurified sIL-1R(I) significantly decreased its thymocyte co-stimulation activity by 57% . Further purification of sIL-1R(I) from other proteins secreted or shed from P . pastoris expression system by size exclusion filtration or ammonium sulfate (60%) precipitation did not influence its capacity to neutralize IL-1 bioactivity . These partially purified sIL-1R(I) preparations significantly decreased thymocyte co-stimulation activity in CM by 70.7 and 77.3%, respectively . In the second experiment, pre-incubation of thymocytes with antisera against the sIL-1R(I) decreased IL-1 activity in CM by 70% relative to control thymocyte cultures that received no antibody and by 59% relative to thymocyte cultures incubated with pre-immune sera . Presumably anti-sIL-1R(I) diminished the IL-1 bioactivity in CM by blocking IL-1 binding to its type-I receptor on thymocytes . Thus, 30% of the IL-1-like activity released by LPS-stimulated HD11 macrophages is probably due to at least one other cytokine . Our data are consistent with the type-I receptor being the primary IL-1 receptor on chicken thymocytes that is capable of providing a signal for proliferation.

Brain Res, 2001 Mar 9, 894(1), 46 - 55
Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils; Yoshida Y et al.; Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct . To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils . MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult . The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult . MK administration at 2 h after occlusion did not ameliorate the neuronal death . These findings suggested that the therapeutic time window was narrow . The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone . These findings suggested that the therapeutic time window was relatively narrow . The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1176 - 82
Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase; Warsame A et al.; Protein disulphide isomerases belong to the thioredoxin superfamily of protein-thiol oxidoreductases that have two double-cysteine redox-active sites and take part in protein folding in the endoplasmic reticulum (ER) . We report here the cloning of a Pichia pastoris genomic DNA fragment (2919 bp) that encodes the full length of a protein disulphide isomerase (PpPDI) . The deduced amino acid sequence of PDI consists of 517 residues and carries the two characteristic PDI-type redox-active domains -CGHC-, separated by 338 residues, and two potential N-glycosylation sites . The N-terminal end forms a putative signal sequence, and an acidic C-terminal region represents a possible calcium-binding domain . Together with the -HDEL ER retrieval sequence at the C-terminus, these features indicate that the gene encodes a redox-active ER-resident protein disulphide isomerase . The nucleotide sequence, which also contains two other open reading frames, has been submitted to the EMBL Nucleotide Sequence Database, Accession No . AJ302014 .

Prostate, 2001 Mar 1, 46(4), 298 - 306
Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast); Habeck LL et al.; BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium . Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease . It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1 . To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein . Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide . We describe the expression of active recombinant mature PSA in yeast . METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA . The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor . RESULTS: We report the production of active, mature rPSA in Pichia pastoris . Two forms of rPSA varying slightly in glycosylation were identified . The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate . CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis . In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing .

J Inorg Biochem, 2001 Jan 15, 83(2-3), 193 - 204
Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris; Kucha JA et al.; Lysyl oxidase from Pichia pastoris has been successfully overexpressed . EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively . Lysyl oxidase from P . pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source) . This enzyme also has a relatively broad substrate specificity compared to other amine oxidases . Molecular modeling data suggest that the substrate channel in lysyl oxidase from P . pastoris permits greater active site access than observed in structurally-characterized amine oxidases . This larger channel may account for the diversity of substrates that are turned over by this enzyme.

Appl Microbiol Biotechnol, 2001 Jan, 55(1), 76 - 80
Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes; Govinden R et al.; The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation . Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates . Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted . The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima . Xylitol production was most rapid when the co-substrate was still present . Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate.

Genetika, 2001 Jan, 37(1), 54 - 65
{Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes}; Tarutina MG et al.; Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes . Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size . Nontransforming genes (P-ADE5 and H-LEU2) transform P . methanolica cells at an extremely low rate even when they are combined with transforming genes . The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.

Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 96 - 9
{Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources}; Leonovich SA et al.; Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1 . The mutation mth1 of P . methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity . beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions . The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.

Zhonghua Xue Ye Xue Za Zhi, 2000 Nov, 21(11), 595 - 9
{Expression of recombinant human soluble Flt3 ligand and its in vitro effects on malignant hematopoietic cells}; Xu Z et al.; OBJECTIVE: To express human soluble Flt3 ligand in a yeast expression system . Pichia pastoris, and investigate the effects of recombinant human soluble Flt3 ligand (rhFL) on the proliferation of malignant hematopoietic cells(MHC) and the effect of dexamethasone (DXM) on the expression of Flt3 receptor (Flt3R) and the rhFL induced proliferation of those cells . METHODS: An artificial gene for rhFL was synthesized by using favored genetic codons of the yeast Pichia pastoris . Flt3R was determined by flow cytometry . Proliferation of MHCs was measured by microculture tetrazolium (MTT) assay . RESULTS: 1 . An expression vector, pPICZ alpha A-FL, was constructed and electroporated into Pichia pastoris . A yield of over 30 mg/L of yeast culture supernatant was obtained . The rhFL could stimulate the proliferation of Raji and HL-60 cells at concentrations of 10-100 ng/ml, but did not for most of other MHCs . 2 . Flt3R was expressed in 5 of 18 MHC lines and the expression in leukemic blast cells was widespread and extremely heterogeneous . 3 . The presence of the receptor on the surface of MHC did not necessarily imply a significant ligand-induced response, at least in terms of proliferation . By contrast, some Flt3R-negative MHC responded to rhFL . 4 . DXM down-regulated the expression of Flt3R and inhibited the proliferation induced by rhFL in some MHC lines and fresh leukemia cell samples . CONCLUSION: A yield of 30 mg/L of rhFL was obtained . rhFL stimulated the proliferation of Raji, HL-60 cells and some fresh leukemia cells, which could be inhibited by DXM through down-regulation of Flt3R . A combination of FL and DXM might possibly serve to control hematopoietic defects in malignant hematopoietic diseases.

Gene, 2001 Jan 24, 263(1-2), 159 - 69
New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris; Lin Cereghino GP et al.; We describe the isolation and characterization of three new biosynthetic genes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris . The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate decarboxylase, respectively . Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P . pastoris . In addition, we have constructed a series of host strains containing all possible combinations of ade1, arg4, his4, and ura3 auxotrophies to be used with these new vectors.

J Biotechnol, 2001 Mar 9, 86(1), 59 - 70
Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris; Minning S et al.; The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity . Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris . Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level . Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures . One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant . Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed . This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.

Carbohydr Res, 2001 Jan 15, 330(1), 73 - 81
Expression and action pattern of Botryotinia fuckeliana (Botrytis cinerea) rhamnogalacturonan hydrolase in Pichia pastoris; Fu J et al.; The cDNA sequence coding for the complete rhamnogalacturonan hydrolase (RGase) of Botryotinia fuckeliana (Botrytis cinerea) was introduced into Pichia pastoris and expressed under the control of the alcohol oxidase promoter . The RGase was secreted into the medium of the yeast driven by the alpha-factor secretion peptide and could be purified using the C-terminal His6-tag fusion . RGase activity was measured using a traditional reducing end assay with linseed rhamnogalacturonan (RG) as the substrate, or with an assay using a fluorescent RG oligomer as the substrate and detection and identification of hydrolysis products by capillary zone electrophoresis (CZE) . Both methods showed the recombinant enzyme to have a specific activity of about ten units per milligram of protein . Since the CZE method allows identification of the hydrolysis products, it was used to show that the RGase lacks a multiple attack mechanism and needs at least five GalA-Rha repeating disaccharides to be active . This finding is contrary to the action pattern of the native RGase of Aspergillus aculeatus which has the same substrate length requirement, but exhibits multiple attack, leading to products containing only two and three Rha-GalA repeat units without the appearance of intermediate sized fragments . No plant cell wall degrading enzymes were detected in the culture medium of un-transformed P . pastoris, thus the recombinant enzyme, devoid of extraneous activities, can be applied for fine structural studies on cell walls.

Cell Mol Life Sci, 1999 Dec, 56(11-12), 1020 - 47
Are elicitins cryptograms in plant-Oomycete communications?
Ponchet M, Panabieres F, Milat M-L, Mikes V, Montillet JL, Suty L, Triantaphylides C, Tirilly Y, Blein JP.
Stimulation of plant natural defenses is an important challenge in phytoprotection prospects . In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants . Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance . However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin . The understanding of factors involved in this reactivity is needed to develop agronomic applications . In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane . Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction . The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies . The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity . This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants . Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel . Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants . Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control . Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed . A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

J Immunother, 2001 Jan-Feb, 24(1), 27 - 36
Engineering and characterization of a novel fusion protein incorporating B7.2 and an anti-ErbB-2 single-chain antibody fragment for the activation of Jurkat T cells; Marshall KW et al.; The provision of the T-cell costimulatory molecule B7 to tumor cells can be an effective way to trigger a tumor-specific cytolytic T-cell response . One way to provide B7 to tumor cells would be to couple an antitumor antibody directly to B7 . Such a molecule should target tumors displaying antigen and provide the costimulatory signal to T cells, resulting in the initiation of an antitumor T-cell response . To this end, a fusion protein was designed that incorporates a single-chain antibody fragment (scFv) to erbB-2 (Her2/neu), an oncogene product overexpressed by 30% to 50% of breast carcinomas, and the ECD of B7-2 (CD86) . This fusion protein, expressed and purified from Pichia pastoris, was shown to retain binding activity to both counter receptors, erbB-2 and CD28 . The fusion protein was also shown to target erbB-2-positive tumor cells and to deliver a CD28-specific T-cell costimulatory signal . These results suggest that a fusion protein engineered to target tumor cells and signal T cells for activation may be an effective means of cancer immunotherapy . Further studies should be performed to characterize the fusion protein in erbB-2 tumor-bearing mice for in vivo tumor targeting, biodistribution, and efficacy.

J Dairy Sci, 2001 Jan, 84(1), 44 - 9
Cation-exchange purification of mutagenized bovine beta-casein expressed in transgenic mouse milk: its putative Asn68-linked glycan is heterogeneous; Choi BK et al.; Bovine beta-casein (A2 genetic variant) was mutagenized to (L70S/P71S) and expressed in transgenic mouse milk . This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X-S/T) (3) . Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it . This novel protein was purified from transgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatography (FPLC) . The novel beta-casein was separated without cross contamination from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2-beta-mercaptoethanol . The purified (L70S/P71S) beta-casein showed an N-linked oligosaccharide attached to Asn68 and different lectin binding profiles compared with the same protein expressed in yeast . The mouse-expressed beta-casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated beta-casein expressed in Pichia pastoris (4) . In addition, the five serine residues located at amino terminus of wild type bovine beta-casein were shown to be normally phosphorylated as in native bovine beta-casein.

Biotechniques, 2001 Jan, 30(1), 81 - 4, 86
Acetylcholinesterase assay for rapid expression screening in liquid and solid media; Villatte F et al.; The synaptic enzyme acetylcholinesterase (AChE), which is the target of many insecticides and potential warfare agents, is implied in Alzheimer's disease and is a good potential candidate to be used in biosensors . This promotes a strong demand for production of recombinant AChE to be used in various studies . A promising expression system is the yeast Pichia pastoris, but the expression efficiency needs to be improved . Optimization studies require a rapid and efficient screening test to detect positive yeast colonies after transformation . Using indoxylacetate as a substrate, we designed a chromogenic test that is not interfered with by the culture media background color and, thus, is suitable for microplate screening . Moreover, it was possible to adapt the test for direct on-plate detection of AChE-expressing colonies.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2437 - 44
Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization; Chatani E et al.; A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion . Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated . After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture . N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A . Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B . Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C . The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc . The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.

Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 631 - 5
{Metabolic calculation of the growth phase in rHSA fermentation}; Huang MZ et al.; The model equations of the growth phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance . The unknown parameters of the model were estimated by multivariable optimization . The model can preferably describe the relations between different macroscopic reaction rates of the process and provide the key for the high-density cultivation of Pichia pastoris.

Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 561 - 5
{Expression of human GDNF in methyltrophic yeast Pichia pastoris and silkworm larvae}; Chen ZY et al.; The cDNA encoding glial cell derived neurotrophic factor (GDNF) was cloned into the Pichia expression vector pPIC9K and then transformed into his4 mutant yeast GS115 by electroporation . Multicopy transformants were screened by various G418 concentrations and induced by methanol . The human GDNF gene was cloned into the baculovirus transfer vector pBacPAK8 . The recombinant transfer vector pBacPAK-GDNF was coinfected with linear Bm-BacPAK6 DNA into BmN cells . The recombinant virus was screened and plaque-purified . The silkworm larvae were infected with the recombinant virus and collected 5 days later . SDS-PAGE and Western blot confirmed that GDNF was expressed in Pichia culture medium and silkworm larvae hemolymph . The GDNF protein expressed in Pichia and silkworm larvae could significantly promote the survival and neurite outgrowth of dopaminergic neurons.

Genetika, 2000 Dec, 36(12), 1634 - 44
{Genetic control of purine biosynthesis in Pichia methanolica yeast . The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase}; Dutova TA et al.; By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene) . This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group . Study of characteristics of spontaneous mutations in early BPN genes of P . methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene . Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene . The DNA sequence that can compensate for the P . methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P . methanolica by the ade6 mutation complementation in the recipient S . cerevisiae strain.

Int, J . Angiol. . 2001 Jan, 10(1), 45 - 46
Candida Tricuspid Endocarditis in a Non-addict; Naseri E et al.; Isolated native non-rheumatic fungal tricuspid valve endocarditis is rarely described in the absence of intravenous drug addiction or use of intracardiac catheters or concomitant cardiac anomalies . Herein, we report a case of tricuspid valve endocarditis in a non-addict, which was successfully treated with valve replacement . The cultures of blood and vegetations revealed Candida Pichia Etschelsii . Candida tricuspid endocarditis must be considered in any patient with tricuspid vegetation, regardless of predisposing factors . </hea

Biotechnol Appl Biochem, 2001 Feb, 33(Pt 1), 35 - 45
Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation; Letourneur O et al.; A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris . The truncated protein lacked the C-terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site . The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography . Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa . This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants . Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses . The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation . However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P . pastoris could be recognized as antigenic structures . As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent . Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P . pastoris is a potential drawback that should be considered when using this expression system.

J Agric Food Chem, 2001 Jan, 49(1), 183 - 8
Antimicrobial effect of extracts from Chinese chive, cinnamon, and corni fructus; Mau J et al.; Extracts were prepared from Chinese chive (Allium tuberosum), cinnamon (Cinnamomum cassia), and corni fructus (Cornus officinalis) and used to evaluate their antimicrobial activity on common foodborne microorganisms, alone and in combination . The mixed extract, consisting of three extracts in equal volumes, showed an entire antimicrobial spectrum and had excellent stability to heat, pH, and storage . The mixed extract exhibited better inhibition on growth of Escherichia coli than potassium sorbate at 2-5 mg/mL . The mixed extract inhibited the growth of Pichia membranaefaciens at levels as low as 2 mg/mL . When the mixed extract was used in foods, the expected antimicrobial effect in orange juice, pork, and milk was observed . After gel filtration chromatography, each extract was partially purified into fractions, and one fraction in each extract showed enhanced antimicrobial activity . Overall, the mixed extract was of promising potential for incorporation into various food products for which a natural antimicrobial additive is desired.

Microsc Res Tech, 2000 Dec 15, 51(6), 563 - 72
Structure and function of the yeast vacuole and its role in autophagy; Thumm M; The vacuole of the yeast Saccharomyces cerevisiae plays an important role in pH- and ion-homeostasis, and is used as a storage compartment for ions . Another important function of the vacuole, especially during nutrient limitation, is the bulk degradation of proteins and even whole organelles . To carry these proteins into the vacuolar lumen, sophisticated transport pathways have evolved . In this review, starvation-induced autophagy and its relationship to the specific cytoplasm to vacuole targeting (cvt-) pathway of proaminopeptidase I is discussed . A further topic is the specific vacuolar uptake and degradation of peroxisomes in Pichia pastoris cells via micro- and macroautophagy .

J Appl Microbiol, 2001 Feb, 90(2), 248 - 55
Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli; Basaran P et al.; AIMS: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543 . METHODS AND RESULTS: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan . All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping . The nucleotide sequence of the P . stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da . The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites . The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively . CONCLUSION: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail . SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to develop better xylanase-utilizing yeast strains.

J Appl Microbiol, 2001 Feb, 90(2), 208 - 15
Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate; Nigam JN; AIMS: The objective of this study was to develop a mutant from Pichia stipitis NRRL Y-7124, tolerant of high concentrations of acetic acid and other inhibitory components present in acid hydrolysates, to improve ethanol yield and productivity . METHODS AND RESULTS: The mutant was developed through adaptation in acid hydrolysate supplemented with nutrients and minerals at 30 +/- 0.5 degrees C . When it was tested for its ability to ferment acid hydrolysate, it showed shorter fermentation time, better tolerance to acid and could ferment at lower pH . The ethanol yield (Yp/s) and productivity (Qp) were increased 1.6- and 2.1-fold, respectively . CONCLUSION: The development of a mutant and its tolerance to acetic acid present in hydrolysates is described . The selected mutant is capable of fermenting both hexoses and pentoses present in hydrolysate at lower pH in comparison with the parent strain . SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant could play a significant role in reducing environmental pollution by using sugars present in pulp mill effluent and, at the same time, could produce a marketable liquid fuel ethanol.

Eur J Biochem, 2001 Feb, 268(3), 752 - 60
Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.); Briand L et al.; In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs) . We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris . ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing . The pairing {Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)} was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs . CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs . Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent . A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2 . Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all . The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP . These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable . At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.

Clin Exp Allergy, 2001 Jan, 31(1), 116 - 24
Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding; Yasuhara T et al.; BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens . Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT) . Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed . OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1 . METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris . Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively . RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium . Their prosequences were removed autocatalytically by dialysis against acidic buffer . Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE . Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities . CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy . Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.

Vaccine, 2001 Feb 8, 19(13-14), 1616 - 27
Generation of a recombinant chimeric Newcastle disease virus vaccine that allows serological differentiation between vaccinated and infected animals; Peeters BP et al.; Using a recently developed reverse genetics system, we have generated a recombinant Newcastle disease virus (NDV) vaccine in which the gene encoding the hemagglutinin-neuraminidase (HN) has been replaced by a hybrid HN gene consisting of the cytoplasmic domain, transmembrane region, and stalk region of HN of NDV, and the immunogenic globular domain of HN of avian paramyxovirus type 4 (APMV4) . The objective was to generate a chimeric live vaccine that induces a protective immune response against NDV by eliciting neutralizing antibodies against the fusion (F) protein, but which can be differentiated from wild-type NDV on the basis of different antibodies elicited by their HN proteins . Pathogenicity tests in day-old chickens showed that the recombinant was non-virulent (intracerebral pathogenicity index {ICPI}=0.00) . A vaccination-challenge experiment in 4-week-old specific pathogen free chickens demonstrated that the recombinant was completely safe and was able to protect chickens from challenge with a lethal dose of virulent NDV . By using a secreted form of HN produced in Pichia pastoris, a test was developed that allowed serological differentiation between animals vaccinated with the recombinant vaccine and animals infected with NDV . These results demonstrate that genetically modified marker vaccines can be generated from small RNA viruses that lack non-essential genes.

Enzyme Microb Technol, 2001 Feb 1, 28(2-3), 139 - 144
Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris; Trujillo LE et al.; Levansucrase (LsdA) (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) yields high levels of fructo-oligosaccharides (FOS) from sucrose . A DNA fragment encoding the precursor LsdA lacking the first 57 amino acids was fused to the pho1 signal sequence under the control of the Pichia pastoris-alcohol oxidase 1 (AOX1) promoter . Methanol induction of a P . pastoris strain harboring a single copy of the lsdA expression cassette integrated in the genome resulted in the production of active levansucrase . After fermentation of the recombinant yeast, LsdA activity was detected in the periplasmic fraction (81%) and in the culture supernatant (18%) with an overall yield of 1% of total protein . The recombinant LsdA was glycosylated and displayed optimal pH and temperature for enzyme activity similar to those of the native enzyme, but thermal stability was increased . Neither fructosylpolymerase activity nor FOS production was affected . Incubation of recombinant LsdA in sucrose (500 g l(-1)) yielded 43% (w/w) of total sugar as 1-kestose, with a conversion efficiency about 70% . Intact recombinant yeast cells also converted sucrose to FOS although for a 30% efficiency.

Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 454 - 9
The genes of two G-proteins involved in protein transport in Pichia pastoris; Huynh TT et al.; Members of the Rab protein family play essential roles in vesicle fusion during protein secretion and represent highly conserved GTP binding proteins . The Saccharomyces cerevisiae Sec4p and Ypt1p, promoting vesicle fusion at the plasma membrane and in ER-Golgi transport, respectively, are among the best characterised yeast members . We have here cloned the Pichia pastoris SEC4 homologue using a S . cerevisiae SEC4 probe . In addition we isolated a crosshybridising clone encoding another Rab-/Ypt-like protein . The deduced full-length PpSec4p comprises 204 amino acid residues with an over all identity of 64% to the Sec4p from S . cerevisiae and 72% to the Candida albicans Sec4p . The YPT-like gene encodes a 216 amino acid residue protein showing highest similarity to the S . cerevisiae Ypt10p and Ypt53p . Both PpSec4p and the Ypt-like protein carry a -Cys-Cys C-terminus, indicating that these proteins are targets for geranyl-geranylation by a type II prenyltransferase .

Protein Expr Purif, 2001 Feb, 21(1), 156 - 64
Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris; Marty C et al.; The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis . Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis . We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds . The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents . Plasmid expression, culture conditions, and purification were optimized in 1-L cultures . The scFv antibody fragments were purified by anion exchange chromatography . The yields were 5-20 mg/L culture medium . The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg . The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F . The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells . Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells . Our results indicate that the P . pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments .

Protein Expr Purif, 2001 Feb, 21(1), 141 - 8
Purification and kinetic characterization of CTP:phosphocholine cytidylyltransferase from Saccharomyces cerevisiae; Friesen JA et al.; CTP:phosphocholine cytidylyltransferase (CCT) regulates the biosynthesis of phosphatidylcholine in mammalian cells . In order to understand the mechanism by which this enzyme controls phosphatidylcholine synthesis, we have initiated studies of CCT from the model genetic system, the yeast Saccharomyces cerevisiae . The yeast CCT gene was isolated from genomic DNA using the polymerase chain reaction and was found to encode tyrosine at position 192 instead of histidine, as originally reported . Levels of expression of yeast CCT activity in Escherichia coli or in the yeast, Pichia pastoris, were somewhat low . Expression of yeast CCT in a baculovirus system as a 6x-His-tag fusion protein was higher and was used to purify yeast CCT by a procedure that included delipidation . Kinetic characterization revealed that yeast CCT was activated approximately 20-fold by 20 microM phosphatidylcholine:oleate vesicles, a level 5-fold lower than that necessary for maximal activation of rat CCT . The k(cat) value was 31.3 s(-1) in the presence of lipid and 1.5 s(-1) in the absence of lipid . The K(m) values for the substrates CTP and phosphocholine did not change significantly upon activation by lipids; K(m) values in the presence of lipid were 0.80 mM for phosphocholine and 1.4 mM for CTP while K(m) values in the absence of lipid were 1.2 mM for phosphocholine and 0.8 mM for CTP . Activation of yeast CCT, therefore, appears to be due to an increase in the k(cat) value upon lipid binding .

Protein Expr Purif, 2001 Feb, 21(1), 92 - 8
Human pS2/trefoil factor 1: production and characterization in Pichia pastoris; Kannan R et al.; The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris . hTFF1 was secreted in large amounts in the extracellular medium of P . pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter . The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography . The resulting hTFF1 was found to be intact by Western blot analysis . Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide . Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells .

Protein Expr Purif, 2001 Feb, 21(1), 71 - 80
Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris; Vassileva A et al.; High-level expression and efficient assembly of hepatitis B surface Antigen (HBsAg) has been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under control of the AOX1 promoter . To fully utilize the expression potential of the P . pastoris expression system, we investigated the influence of gene copy number on the expression of HBsAg in this yeast . A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach . Using this strategy, constructs containing up to a maximum of eight direct repeats of the HBsAg-expressing cassettes could be created . These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX1 gene . Deletion of the AOX1 gene was intended to create Mut(s) (methanol utilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form . A systematic investigation of the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the steady-state levels of the HBsAg-specific mRNA, which in turn is closely paralleled by a corresponding increase in the total levels of the HBsAg protein . Virtually all the recombinant protein in the soluble fraction was present in the particulate form based on particle-specific ELISA and sedimentation behavior . Further, our studies also revealed the continued physical and functional integrity of the HBsAg-expressing cassettes during the course of an extended induction phase spanning 6 days .

Protein Expr Purif, 2001 Feb, 21(1), 65 - 70
Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin; Schwettmann L et al.; ADAM 9 is a member of the cellular metalloprotease/disintegrin/cysteine-rich (MDC) gene family, related to soluble snake venom metalloproteases (SVMP) . ADAMs may play important roles in cell-cell fusion, cell-matrix interaction, and other cellular functions . To investigate catalytic activity of human ADAM 9 we have cloned and expressed the metalloprotease domain of human ADAM 9 in Pichia pastoris . The recombinant protein was purified in a three-step purification procedure and activity was detected against gelatin, beta-casein, and fibronectin . In addition we identified five normal and cancer cell lines expressing mRNA of human ADAM 9 .

Protein Expr Purif, 2001 Feb, 21(1), 13 - 23
Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic bacillus alpha-amylase produced in Pichia pastoris; Tull D et al.; Alkalophilic Bacillus alpha-amylase (ABA) was produced in the yeast Pichia pastoris with a yield of 50 mg L(-1) of culture supernatant . The recombinant protein, rABA, was glycosylated at seven of the nine sites for potential N-glycosylation as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments . The number of hexose units within each glycan chain was found to vary from 8 to 18 as calculated from the masses of glycosylated peptide fragments . Temperature stability measurements in the absence of substrate showed that the T(50) of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76 degrees C while that of ABA purified from Bacillus was 89 degrees C thus demonstrating that the original temperature stability of ABA was not retained by rABA . The relative thermoperformance, i.e., the activity at 80 degrees C relative to that at 37 degrees C was 0.9 +/- 0.3 for rABA . Removal of all seven N-linked glycans by endoglycosidase H increased the relative thermoperformance to 2.4 +/- 0.6, compared to the value of 3.5 +/- 1.1 for ABA . Thus, removal of the N-linked glycans did not improve the thermostability of rABA but modified its thermoperformance to approach that of the original Bacillus enzyme . rABA had the highest activity around pH 6 . Treatment of rABA with endoglycosidase H shifted the pH activity profile in a more alkaline direction approaching the pH activity profile of ABA .

Clin Chem, 2001 Feb, 47(2), 237 - 46
Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues; Luo LY et al.; BACKGROUND: The human kallikrein 10 gene {KLK10, also known as normal epithelial cell-specific 1 gene (NES1)} is a member of the human kallikrein gene family . The KLK10 gene encodes for a secreted serine protease (hK10) . We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states . The aim of this study was to develop a sensitive and specific immunoassay for hK10 . METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system . The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera . A sandwich-type immunofluorometric assay was then developed using these antibodies . RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L . The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6) . The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10% . hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum . CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts . This assay can be used to examine the value of hK10 as a disease biomarker.

J Virol, 2000 May, 74(9), 4074 - 84
Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris; Hwang JS et al.; Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome . In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris . Simultaneous expression of all three P proteins in the yeast P . pastoris was achieved by the addition of methanol . To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus . Starting from the induced P . pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography . The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer . The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex . Furthermore, the 3P complex showed capped RNA-primed RNA synthesis . Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P . pastoris.

Cancer Res, 2000 Dec 15, 60(24), 6964 - 71
Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application; Goel A et al.; Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors . Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications . In the present study, we report the generation of a tetravalent single-chain Fv {{sc(Fv)2}2} of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72 . {Sc(Fv)2}2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter . The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore . The binding affinity constant (K(A)) for the {sc(Fv)2}2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv {sc(Fv)2; 2.75 x 10(7) M(-1)} . At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated {sc(Fv)2}2, sc(Fv)2, and IgG, respectively . Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for {sc(Fv)2}2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively . The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Plant Physiol, 2001 Jan, 125(1), 378 - 86
Molecular and biochemical characterization of a cytokinin oxidase from maize; Bilyeu KD et al.; It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta . Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance . Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented . Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme . The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals . Expression of the oxidase in maize tissues is described.

FEBS Lett, 2000 Dec 22, 487(1), 87 - 90
Genomic exploration of the hemiascomycetous yeasts: 15 . Pichia sorbitophila; de Montigny J et al.; This paper reports the genomic analysis of the strain CBS7064 of Pichia sorbitophila, a homothallic diploid yeast . We sequenced 4829 random sequence tags of about 1 kb and compared them to the Saccharomyces cerevisiae gene products . Approximately 1300 nuclear genes, 22 tRNAs, the rDNA locus, and six mitochondrial genes have been identified . The analysis of the rDNA genes has permitted to classify this organism close to the Candida species . Accession numbers from AL414896 to AL419724 at EMBL databank.

FEBS Lett, 2000 Dec 22, 487(1), 76 - 81
Genomic exploration of the hemiascomycetous yeasts: 13 . Pichia angusta; Blandin G et al.; As part of a comparative genomics project on 13 hemiascomycetous yeasts, the Pichia angusta type strain was studied using a partial random sequencing strategy . With coverage of 0.5 genome equivalents, about 2500 novel protein-coding genes were identified, probably corresponding to more than half of the P . angusta protein-coding genes, 6% of which do not have homologs in Saccharomyces cerevisiae . Some of them contain one or two introns, on average three times shorter than those in S . cerevisiae . We also identified 28 tRNA genes, a few retrotransposons similar to Ty5 of S . cerevisiae, solo long terminal repeats, the whole ribosomal DNA cluster, and segments of mitochondrial DNA . The P . angusta sequences were deposited in EMBL under the accession numbers AL430961 to AL436044.

Appl Microbiol Biotechnol, 2000 Dec, 54(6), 741 - 50
Heterologous protein production in methylotrophic yeasts; Gellissen G; The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression . They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes . An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts . The well-established P . pastoris and H . polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes . Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future . The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past.

Thromb Res, 2000 Dec 1, 100(5), 461 - 7
Catalytic and fibrinolytic properties of recombinant urokinase plasminogen activator from E . coli, mammalian, and yeast cells; Wang P et al.; The enzymatic and fibrinolytic properties of glycosylated and nonglycosylated recombinant human pro-urokinase (pro-UK) produced in yeast Pichia pastoris were characterized and compared with those of Escherichia coli and mammalian cell-derived pro-UK . Among the five different forms of pro-UK, the yeast glycosylated pro-UK was activated by plasmin with the lowest catalytic efficiency (kcat/Km) . The yeast glycosylated urokinase (UK) also had the highest Km in its activation of Glu-plasminogen, and had a substantially lower fibrinolytic activity than the other four forms . These findings suggest that the poly-mannose on Asn-302 of yeast glycosylated pro-UK interfered with its activation by plasmin and its binding interaction with plasminogen . By contrast to plasminogen, the activation of the small synthetic substrate, S2444, was comparable for all five forms of recombinant UK . It is concluded that the glycosyl residue on pro-UK/UK is functionally important and modulates its activatability and its catalytic efficiency against its natural substrate . Therefore, pro-UK from different expression systems cannot be assumed to have comparable fibrinolytic activities.

Vet Immunol Immunopathol, 2000 Dec 29, 77(3-4), 233 - 41
Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I; Mwangi SM et al.; A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity . A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia . Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography . In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml . Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2179 - 85
Purification and characterization of an alpha-L-rhamnosidase from Pichia angusta X349; Yanai T et al.; An intracellular alpha-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps . The alpha-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa . The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 and at around 40 degrees C . The Ki for L-rhamnose inhibition was 25 mM . The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate . The alpha-L-rhamnosidase was highly specific for alpha-L-rhamnopyranoside and liberated rhamnose from naringin, rutin, hesperidin, and 3-quercitrin . The alpha-L-rhamnosidase was active at the ethanol concentrations of wine . It efficiently released monoterpenols, such as linalool and geraniol, from an aroma precursor extracted from Muscat grape juice.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2138 - 44
Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris; Takahashi K et al.; Binding of allergen-IgE complexes to the high affinity IgE receptor (Fc epsilonRI) on mast cells and basophils leads to the release of various mediaters such as histamine . Fab fragments prepared by the papain digestion of humanized antibody against human Fc epsilonRI inhibited the release of histamine from human basophils . Here we established an expression system to directly produce Fab fragments of the humanized anti-human Fc epsilonRI antibody in methylotropic yeast, P . pastoris . Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P . pastoris phosphatase gene . They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides . The obtained Fab fragments inhibited the binding between IgE and Fc epsilonRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.

Yeast, 2001 Jan 15, 18(1), 61 - 7
Cloning and sequence of the LYS2 homologue gene from the osmotolerant yeast Pichia sorbitophila; Bleykasten-Grosshans C et al.; We have isolated the Pichia sorbitophila LYS2 (PsLYS2) gene by complementation of a lys2 Saccharomyces cerevisiae mutant . The sequenced DNA fragment contains a putative ORF of 4197 bp and the deduced translation product shares a global identity of 66% and 58% to the Lys2 protein homologues of Candida albicans and S . cerevisiae, respectively . Analysis of PsLYS2 sequence suggests that, similarly to S . cerevisiae LYS2, it codes for a polypeptide having two separate enzymatic activities which reside in different domains of the protein, including an adenylate domain, an acyl-carrier site and a short-chain reductase domain . Several GCN4- and NIT2-binding motifs have been matched in the promotor sequence of PsLYS2 . In addition, upstream of the sequenced PsLYS2 sequence, we have found the 3'-terminal half of a gene of same orientation encoding a RAD16-like protein, a genomic organization similar to that of C . albicans.

Yeast, 2001 Jan 15, 18(1), 49 - 60
The ALS5 gene of Candida albicans and analysis of the Als5p N-terminal domain; Hoyer LL et al.; ALS genes of Candida albicans encode a family of cell-surface glycoproteins with a three-domain structure . Each Als protein has a relatively conserved N-terminal domain, a central domain consisting of a tandemly repeated motif, and a serine-threonine-rich C-terminal domain that is relatively variable across the family . The ALS family exhibits several types of variability that indicate the importance of considering strain and allelic differences when studying ALS genes and their encoded proteins . Analysis of ALS5 provided additional evidence of variability within the ALS family . Comparison of the ALS5 sequence from two strains indicated sequence differences larger than strain or allelic mismatches observed for other C . albicans genes . Screening a collection of commonly used C . albicans strains and clinical isolates indicated that ALS5 is not present in several of these strains, supporting the conclusion that the Als protein profile is variable among C . albicans isolates . Physical mapping of ALS5 showed that it is located close to ALS1 on chromosome 6 . The N-terminal domain of Als5p was produced in Pichia pastoris to initiate structural analysis of this portion of the protein . The hydrophobic character of this portion of the protein was exploited in the purification scheme . Circular dichroism analysis of the purified, authenticated protein yielded a high content of antiparallel beta-sheet and little to no alpha-helical structure . These results are consistent with the conclusion that the N-terminal domain of Als5p has an immunoglobulin fold structure similar to that found in many cell adhesion molecules . Gene sequences of C . albicans ALS5 (Accession No . AF068866) and TPI1 (Accession No . AF124845) have been deposited in the GenBank database .

Biotechnol Appl Biochem, 2000 Dec, 32 ( Pt 3), 179 - 87
Characterization of recombinant invertase expressed in methylotrophic yeasts; Acosta N et al.; We studied, for the first time, characterization of the invertase expressed in the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris in terms of enzyme conformational stability and structural behaviour induced by temperature as a function of pH using enzymic assays, differential scanning calorimetry, fluorescence and CD . The enzyme produced in both hosts was very stable over a broad range of pH values, keeping its enzymic activity and structure above 60 degrees C . Thermal denaturation, as measured by differential scanning calorimetry, was always irreversible . However, the fact that scanning rate had no effect on the calorimetric curves gave us the chance to analyse the data from a thermodynamic point of view . The conformational stabilities were essentially identical under the experimental conditions studied, but stability was always slightly higher in the enzyme expressed in H . polymorpha . This fact indicates that the greater degree of glycosylation of this enzyme form contributed to its increased global stability . Reactivation upon heating at 80 degrees C depends on protein concentration, suggesting that irreversibility could be associated with slow refolding kinetics at high protein concentration.

Can J Microbiol, 2000 Nov, 46(11), 967 - 80
The use of anonymous DNA markers in assessing worldwide relatedness in the yeast species Pichia kluyveri Bedford and Kudrjavzev; Ganter PF et al.; Pichia kluyveri, a sexual ascomycetous yeast from cactus necroses and acidic fruit, is divided into three varieties . We used physiological, RAPD, and AFLP data to compare 46 P . kluyveri strains collected worldwide to investigate relationships among varieties . Physiology did not place all strains into described varieties . Although the combined AFLP and RAPD data produced a single most parsimonious tree, separate analysis of AFLP and RAPD data resulted in significantly different trees (by the partition homogeneity test) . We then compared the distribution of strains per band to an expected distribution . This suggested we could separate both the AFLP and RAPD datasets into bands from rapidly and slowly changing DNA regions . When only bands from slowly changing regions (from each dataset) were included in the analysis, both the RAPD and AFLP datasets supported a single tree . This second tree did not differ significantly from the cladogram based on all of the DNA data, which we accepted as the best estimate of the phylogeny of these yeast strains . Based on this phylogeny, we were able to demonstrate the strong influence of geography on the population structure of this yeast, confirm the monophyly of one variety, question the utility of maintaining another variety, and demonstrate that the physiological differences used to separate the varieties did not do so in all cases.

Clin Cancer Res, 2000 Nov, 6(11), 4314 - 22
Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting; Rohrbach F et al.; Efficient T-cell activation requires two signals . The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules . One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface . Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity . However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells . As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells . We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase . A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1-111 (CD86(111)) of the mature protein . CD86(111) produced in the yeast Pichia pastoris and CD86(111) expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors . Bacterially expressed CD86(111)-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.

Curr Biol, 2000 Nov 16, 10(22), 1443 - 6
Chaperones that cure yeast artificial {PSI+} and their prion-specific effects; Kushnirov VV et al.; The {PSI(+)} nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation {1} . Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure {PSI(+)} {2} . Overproduction of the chaperone protein Ssb1 increased the {PSI(+)} curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected {PSI(+)} against Hsp104 {3,4} . We used an artificial {PSI(+)(PS)} based on the Sup35 prion-forming domain from yeast Pichia methanolica {5} to find other prion-curing factors . Both {PSI(+)(PS)} and {PSI(+)} have prion 'strains', differing in their suppressor efficiency and mitotic stability . We show that {PSI(+)(PS)} and a 'weak' strain of {PSI(+)} can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1 . The ability of different chaperones to cure {PSI(+)(PS)} showed significant prion strain specificity, which could be related to variation in Sup35 prion structure . Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.

Mol Biotechnol, 2000 Sep, 16(1), 23 - 52
Recombinant protein expression in Pichia pastoris; Cregg JM et al.; The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein . P . pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool . What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system . To date well over 200 heterologous proteins have been expressed in P . pastoris . Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

J Biochem (Tokyo), 2000 Dec, 128(6), 999 - 1008
Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein; Ketudat Cairns JR et al.; Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides . To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning . The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein . The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme . The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved . Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified . Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry . The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized . The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.

J Biochem (Tokyo), 2000 Dec, 128(6), 891 - 5
Single-chain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization; Andrade EV et al.; We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22 . The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A . The second vector codes for the scFv fused to the Fc fragment of the human IgG1 . The fusion partner simplified the detection and purification of the secreted protein . These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.

Genetika, 2000 Oct, 36(10), 1322 - 9
{Prionization of the Pichia methanolica SUP35 gene product in the yeast Saccharomyces cerevisiae}; Zadorskii SP et al.; Induction of the prionlike form of the SUP35 gene of Pichia methanolica, the {PSIP+} factor, was shown in the transgenic yeast Saccharomyces cerevisiae containing the P . methanolica SUP35 gene located in the chromosome instead of the indigenous SUP35 gene . Either the induction of the {PSIP+} factor in the transgenic yeast, unlike that of the classical {PSI+} factor, does not depend on the presence of the {PIN+} determinant in the cell or the substitution of the S . cerevisiae SUP35 gene for the P . methanolica SUP35 gene changes the PIN status of the strain . The {PSIP+} factor is unstable in mitosis and meiosis and is not effectively eliminated upon over-production of the chaperone protein Hsp104p of S . cerevisiae . The existence of an interspecific barrier during transmission of the prionlike state from S . cerevisiae Sup35p to P . methanolica Sup35p was shown.

Mol Pharmacol, 2000 Dec, 58(6), 1609 - 15
Importance of histidine residues for the function of the human liver UDP-glucuronosyltransferase UGT1A6: evidence for the catalytic role of histidine 370; Ouzzine M et al.; The human UDP-glucuronosyltransferase isoform UGT1A6 catalyzes the nucleophilic attack of phenolic xenobiotics on glucuronic acid, leading to the formation of water-soluble glucuronides . Based on the irreversible inhibition of the enzyme activity by the histidyl-selective reagent diethyl pyrocarbonate (DEPC), histidine was suggested to play a key role in the glucuronidation reaction . Therefore, the role of four strictly conserved histidine residues (His38, His361, His370, and His485) in the glucuronidation of 4-methylumbelliferone, as reporter substrate, was examined using site-directed mutagenesis . For this purpose, stable heterologous expression of wild-type and mutant UGT1A6 was achieved in the yeast Pichia pastoris . Replacement of histidine residues by alanine or glutamine led to fully inactive H38A, H38Q, and H485A mutants . Substitution of His361 by alanine affected the interaction of the enzyme with the cosubstrate, as indicated by a 4-fold increase in the K(m) value toward UDP-glucuronic acid . Interestingly, H370A mutant presented a severely impaired catalytic efficiency (with a V(max) value approximately 5% that of the wild-type), whereas conservative substitution of His370 by glutamine (H370Q) led to a significant restoration of activity . Whereas H361A was inactivated by DEPC as the wild-type enzyme, this chemical reagent only produced a minor effect on either H370Q or H370A mutant, providing evidence that His370 is probably the reactive histidine residue targeted by DEPC . The dramatic changes in catalytic efficiency on substitution of His370 by alanine and the ability of glutamine to function in place of histidine along with a weak sensitivity of these mutants to DEPC strongly suggest that His370 plays a catalytic role in the glucuronidation reaction.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 499 - 509
Cloning, sequencing, and functional analysis of H-OLE1 gene encoding delta9-fatty acid desaturase in Hansenula polymorpha; Lu SF et al.; H-OLE1, a gene encoding delta9-fatty acid desaturase (FAD) in Hansenula polymorpha strain CBS 1976, was isolated by hybridization based upon its homology with the P-OLE1 gene cloned earlier from a related species, Pichia angusta IFO 1475 . The sequence of the H-OLE1 gene revealed high structural conservation with delta9-FADs from various organisms . A putative 451-amino acid polypeptide encoded by the gene, like all other delta9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidine clusters, and a C-terminal cytochrome b5-like domain which has been suggested to be involved in electron transport in desaturation reactions . The whole H-OLE1 gene complemented a H . polymorpha fad1 mutation leading to a defect in delta9-FAD . However, the unsaturated fatty acid requirement that the Saccharomyces cerevisiae ole1 mutant displays was complemented by only the open reading frame of H-OLE1 driven by S . cerevisiae glyceroaldehyde-3-phosphate dehydrogenase promoter, but not by the intact H-OLE1, suggesting that the H . polymorpha delta9-FAD was compatible with the desaturation system of S . cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells . It was shown by Northern hybridization that transcription of the H-OLE1 gene in H . polymorpha was slightly repressed by exogenous delta9-unsaturated fatty acid . An H . polymorpha disruption mutant (deltaH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupted H-OLE1::S-LEU2 linear DNA . It was shown by genetic and molecular analyses that input DNA was integrated in several copies into the chromosomal target to replace the mutated fad1 allele . Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and deltaHOLE1 disruption mutants.

Protein Expr Purif, 2000 Dec, 20(3), 472 - 84
Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris; Chen Y et al.; Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A . This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids . An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy . Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells . In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A . Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods . Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor . Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C . The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation . Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions . Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates .

Protein Expr Purif, 2000 Dec, 20(3), 462 - 71
A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen; Best EA et al.; Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma . Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form . We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors . Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P . pastoris . The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa . Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion . A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P . pastoris as a mature, unglycosylated, approximately 25-kDa protein . The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P . pastoris and to nDer f 1 obtained from mites . Thus, oligosaccharides are not required for secretion from P . pastoris or for IgE binding in vitro . Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels . The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy .

Protein Expr Purif, 2000 Dec, 20(3), 389 - 93
Cloning of Trypanosoma cruzi trans-sialidase and expression in Pichia pastoris; Laroy W et al.; Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface . This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5'-monophospho-N-acetylneuraminic acid as a donor substrate . Also, the common glycosyltransferase structure is not present . To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells . Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris . The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification . Using P . pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium .

Protein Expr Purif, 2000 Dec, 20(3), 372 - 8
A system for dual protein expression in Pichia pastoris and Escherichia coli; Lueking A et al.; We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems . In this vector, an E . coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX) . For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites . A P . pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1) . In the present study, the expression of human proteins in P . pastoris and E . coli was compared using this single expression vector . For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones . After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria . All proteins were expressed soluble in P . pastoris, whereas in E . coli only 31% could be purified under native conditions . Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning .

Biochemistry, 2000 Nov 21, 39(46), 14138 - 49
Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein; Urbatsch IL et al.; Mutagenesis was used to investigate the functional role of six pairs of aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters . Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant . Mutations at other acidic residues had no apparent effect in the same assays . These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity . Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido{alpha-(32)P}nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3 . The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity . Surprisingly, drugs did stimulate vanadate trapping of 8-azido{alpha-(32)P}nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3 . This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed . In addition, photolabeling by 8-azido{alpha-(32)P}nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants . These results suggest that steps after the transition state, possibly involved in release of MgADP, are severely impaired in these mutant enzymes.

Biotechniques, 2000 Nov, 29(5), 1094 - 9
New method for the selection of multicopy transformants of Pichia pastoris, using 3-amino-1,2,4 triazol; Menendez J et al.; The methylotrophic yeast Pichia pastoris has been successfully used for the expression of many heterologous proteins . The level of expression of some of these proteins depends on the copy number of the gene inserted into the yeast genome . Several methods have been reported in the past few years for the isolation of multicopy transformants . One of these methods used an expression vector that contains the bacterial kanamycin-resistance gene Tn903kanr, which confers resistance to G418 . Here, we report a different selection method in a mutant strain of P . pastoris (his3-) based on the resistance to 3-amino-1,2,4 triazol, with a vector containing the HIS3 gene from Saccharomyces cerevisiae . Using this selection method, we isolated here P . pastoris transformants containing several copies of the dextranase gene (dex) from Penicillium minioluteum.

Biotechnol Bioeng, 2001 Jan 5, 72(1), 1 - 11
A rational approach to improving productivity in recombinant Pichia pastoris fermentation; d'Anjou MC et al.; A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity . A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels . The range of dilution rates studied was 0 . 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level) . With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied . A maximum specific methanol consumption rate of 20 mg/g . h was achieved at a dilution rate of 0.06 h(-1) . The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g . h and 1.7 mg/L . h, respectively . Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity . Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources . Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model . Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration . The overall volumetric productivity of both runs was approximately 2.2 mg/L . h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy .

Plant Physiol, 2000 Nov, 124(3), 1217 - 28
Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue; Luscher M et al.; Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone . Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Luscher and Nelson, 1995) . It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases . Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris . When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST . When the cDNA was expressed in P . pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate . The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity . The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Am J Physiol Regul Integr Comp Physiol, 2000 Dec, 279(6), R2026 - 41
Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin; Prapunpoj P et al.; Xenopus laevis transthyretin (xTTR) cDNA was cloned and sequenced . The derived amino acid sequence was very similar to those of other vertebrate transthyretins (TTR) . TTR gene expression was observed during metamorphosis in X . laevis tadpole liver but not in tadpole brain nor adult liver . Recombinant xTTR was synthesized in Pichia pastoris and identified by amino acid sequence, subunit molecular mass, tetramer formation, and binding to retinol-binding protein . Contrary to mammalian xTTRs, the affinity of xTTR was higher for L-triiodothyronine than for L-thyroxine . The regions of the TTR genes coding for the NH(2)-terminal sections of the polypeptide chains of TTR seem to have evolved by stepwise shifts of mRNA splicing sites between exons 1 and 2, resulting in shorter and more hydrophilic NH(2) termini . This may be one molecular mechanism of positive Darwinian evolution . Open reading frames with xTTR-like sequences in the genomes of C . elegans and several microorganisms suggested evolution of the TTR gene from ancestor TTR gene-like "DNA modules." Increasing preference for binding of L-thyroxine over L-triiodothyronine may be associated with evolving tissue-specific regulation of thyroid hormone action by deiodination.

J Cardiovasc Pharmacol, 2000 Nov, 36(5 Suppl 1), S55 - 7
Expression and characterization of the human endothelin-A-receptor in Pichia pastoris: influence of N-terminal epitope tags; Cid GM et al.; Knowledge of the three-dimensional structure of the endothelin-A receptor (ET(A)) will provide important information for rational drug design of antagonists and agonists . In order to produce correctly folded and active receptor protein for physical characterization by such techniques as X-ray crystallography and circular dichroism spectroscopy, we have cloned and expressed the human ET(A)-receptor in Pichia pastoris . The expression constructs all contained signal sequences to direct the expressed protein to the membrane fraction . Fidelity of folding and the subtype specificity of the expressed receptor was demonstrated by ligand binding studies using 125I-labelled endothelin-1 (ET-1) . Expression of receptor with two different N-terminal epitope 'tags' had little effect on the affinity of ET-1 for the receptor . The strain of P . pastoris employed did influence the quantity of receptor expressed.

Mol Immunol, 2000 May, 37(7), 361 - 75
Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1); McDermott MJ et al.; An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively . Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively . A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C . felis salivary gland cDNA library, using a combination of PCR and hybridization screening . This cDNA is 658 bp in length, and contains an open reading frame of 528 bp . The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein . The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively . The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy . Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells . Approximately, 90% of the rCte f 1 expressed in E . coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity . Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E . coli cells . However, P . pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1 . Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen . The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen . Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Nucleic Acids Res, 2000 Nov 15, 28(22), 4479 - 87
Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance; Tomaska L et al.; Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure . The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension . The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown . While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation . Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I . The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7) . Similar results were obtained with two other yeast species (P.philodendra and C . salmanticensis) which possess analogous telomeric structure.

Microbiology, 2000 Nov, 146 ( Pt 11), 2765 - 73
Intra- and intermolecular events direct the propeptide-mediated maturation of the Candida albicans secreted aspartic proteinase Sap1p; Beggah S et al.; Pathogenic yeasts of the genus Candida secrete aspartic proteinases (Sap) which are synthesized as preproenzymes . Expression of the C . albicans SAP1 gene lacking the propeptide-coding region in the methylotrophic yeast Pichia pastoris does not lead to the secretion of the enzyme into the culture supernatant, but results in an accumulation of recombinant protein in the cell . Co-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion . A deletion analysis revealed that only a 12 aa sequence in the propeptide, corresponding to a highly conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant . No Sap1p was secreted when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence . However, the simultaneous production of equivalent amounts of Sap1p and His-tagged Sap1p (H(6)-Sap1p) with a mutated and a non-mutated propeptide, respectively, led to the secretion of both proteins in a ratio of approximately 1:2 . The restoration of Sap1p secretion occurred at the expense of secretion of H(6)-Sap1p since the total activity was comparable to that of strains producing only H(6)-Sap1p with a non-mutated propeptide . In contrast, the proteolytic activity of strains secreting Sap1p and H(6)-Sap1p both with a functional propeptide was twice that of strains producing either Sap1p or H(6)-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant . Altogether, these results show that the propeptide can only function once and that the maturation of recombinant C . albicans secreted aspartic proteinase Sap1p is directed through a combination of intra- and inter-molecular pathways.

Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 308 - 11
{Synonymous codon usage in Pichia pastoris}; Zhao X et al.; According to the synonymous codons used in 28 open reading frames from Pichia pastoris, the codon usage in this species was calculated and 19 codons have been inferred to be its optimal codons . The results show that pattern of the codon usage in P . pastoris is similar to that in S . cerevisiae and in K . lactis except for the synonymous codon of glutamic acid, which may be the special bias of P . pastoris.

Life Sci, 2000 Sep 15, 67(17), 2103 - 15
Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone; Chen CM et al.; In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine . A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide . After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein . Over 95% of rGH was shown to be export into the culture supernatant . Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran . Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks . The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage . The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group . These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

J Biochem (Tokyo), 2000 Nov, 128(5), 823 - 6
An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis; Asami Y et al.; Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal . The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine . However, more than half of the product receives yeast specific mannosylations . The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine . In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis . HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues . Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Appl Environ Microbiol, 2000 Nov, 66(11), 4940 - 4
Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts; Sagt CM et al.; Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility . We used N-glycosylation as a tool to enhance protein secretion . Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S . cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site . When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold . If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold . These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Yeast, 2000 Nov, 16(15), 1387 - 96
Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate; Nakakubo H et al.; We have previously reported efficient production of mature human chymase (h-chymase) using an original system of expression in Pichia pastoris (Nakakubo et al., 2000), whereby recombinant h-chymase (rh-chymase) was secreted as a mature form with the correct N-terminal amino acid sequence and was easily purified . In the course of investigation of secretory rh-chymase, we also found large amounts of chymase to be present in insoluble form in the transformant cell . Although the cellular rh-chymase had no proteolytic activity, its chymotryptic activity was restored in a reconstitution process utilizing guanidine and glutathione . As with secretory rh-chymase, efficient purification was possible by heparin affinity chromatography . The purified cellular rh-chymase showed the same mobility as secretory rh-chymase in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after deglycosylation . N-terminal amino acid sequence analysis revealed that the signal peptide had been correctly removed . K(m) value (5.93 mM), as well as pH profile and inhibition profile toward protease inhibitors of reconstituted cellular rh-chymase, indicated that the rh-chymase enzymatically closely resembles native h-chymase . Furthermore, it showed a greatly restricted proteolytic activity towards Ang I, and formed Ang II without the further cleavage which is a feature of h-chymase . It was thus found that the insoluble rh-chymase stored in the cells could be solubilized and reconstituted to give the same structure as h-chymase, not only in terms of enzyme active site but also of substrate recognition site .

Yeast, 2000 Nov, 16(15), 1377 - 85
Organization of specific genomic regions of Zygosaccharomyces rouxii and Pichia sorbitophila: comparison with Saccharomyces cerevisiae; Sychrova H et al.; The genomes of Zygosaccharomyces rouxii and Pichia sorbitophila were partially explored . The genome of Z . rouxii CBS 732 consists of seven chromosomes with an approximate size of 1.0-2.75 Mb, 12.8 Mb in total . Five of the chromosomes were labelled with specific probes . Three Z . rouxii genomic DNA fragments were sequenced; all 10 ORFs found were without introns and they have homologues in S . cerevisiae . Gene order comparison revealed that the organization is partially conserved in both species . The genome of P . sorbitophila CBS 7064 consists of seven chromosomes with an approximate size of 1.0-2.9 Mb, 13.9 Mb in total . Three of the chromosomes were labelled with specific probes . The sequencing of a 5.2 kb genomic DNA fragment revealed three ORFs, but no conservation of their organization was found, although all of them have their respective homologues in S . cerevisiae . According to our results, the presence of two overlapping ORFs in S . cerevisiae (YJL107c-YJL108c) could be interpreted as the result of a frameshift mutation.

Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 531 - 3
{High-level expression of human vascular endothelial growth factor in Pichia pastoris}; Yan XY et al.; The gene of VEGF165 was subcloned into the P . pastoris secretive expression vector pHIL-S1 and the recombinant expression plasmid pHIL-S1-VEGF165 was constructed . After transformation into yeast GS115, the positive transformants were obtained through phenotype selection and DNA Dot blotting . After induction by methanol, soluble dimer VEGF165 were expressed and secreted into the culture supernatant with its expression occupying 47% of the total protein in the supernatant . Dot blot analysis showed that the expressed human VEGF165 could bind to its receptors flt-1 and KDR.

Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 447 - 50
{Expression and characterization of human vascular endothelial growth factor receptor Flt-1 extracellular domain in Pichia pastoris}; Ma L et al.; By inserting VEGF receptor Flt-1(1-3 loop) cDNA into Pichia pastoris expression vector pPIC9K containing AOX1 promotor and the sequences of alpha secreting signal peptides, the expression plasmid pPIC9K/Flt-1(1-3) was constructed and transformed into GS115 . The multi-copy insert transformants were selected and cultivated in flasks . After 4 days of 1% methanol induction, the expressed Flt-1(1-3) accumulated up to 30% of total proteins in supernatant . The expressed Flt-1(1-3) was further proved with good antigenicity and high specificity by ELISA and Western blot . They can bind to VEGF and inhibit HUVEC proliferation stimulated by VEGF.

Yi Chuan Xue Bao, 2000, 27(7), 641 - 6
{Isolation and characterization of PAOX2 mutant in Pichia pastoris}; Dai XY et al.; Spontaneous Mut+ mutants of P . pastoris AOX1-defective expression strain have been isolated, they were identified as phenotypically utilized methanol to grow as wild type . The results obtained from measuring growth curve when cultivated in medium in which methanol as a sole carbon source and detecting HSA protein on SDS-PAGE confirmed that the mutants have increased ability to utilize methanol and express foreign HSA gene product . The promoter region of AOX2 gene from the mutants has been cloned by PCR amplification, and the DNA fragment is 1022bp in size . Sequencing analysis showed that there are two point mutations at positions of -529 and -255 from the translation initiation codon respectively . The mutations improved AOX-1 defective function and facilitate the foreign gene for higher expression.

Arch Biochem Biophys, 2000 Oct 1, 382(1), 105 - 12
Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris; Rodriguez E et al.; Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties . Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively . Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA . The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%) . Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties . First, it was more active at pH 3.5-5.5 . Second, it retained more (P < 0.01) phytase activity than that of r-AppA . Third, its specific activity of phytase was 54% higher . Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively . Based on the recently published E . coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein . This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.

Protein Expr Purif, 2000 Nov, 20(2), 334 - 45
High-level expression of human liver monoamine oxidase B in Pichia pastoris; Newton-Vinson P et al.; The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described . A 2-L culture of P . pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate . By a modification of the published procedure for purification of bovine liver MAO B {Salach, J . I . (1979) Arch . Biochem . Biophys . 192, 128-137}, recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture) . The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD . One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide . Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini . The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM . Kinetic isotope effect parameters using {alpha,alpha-(2)H(2)}benzylamine are also similar to those found for the bovine enzyme . Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D){k(cat)/K(m)(benzylamine)} = 4.5, and a (D){k(cat)/K(m)(O(2))} = 1.0 . In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme . These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible .

Protein Expr Purif, 2000 Nov, 20(2), 216 - 27
Large-scale purification of recombinant human angiostatin; Shepard SR et al.; A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported . rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography . Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification . Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis . On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9) . The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials . Methods development, process synthesis, and process scale-up data are presented and discussed .

Protein Expr Purif, 2000 Nov, 20(2), 207 - 15
Expression and purification of mammalian calreticulin in Pichia pastoris; Andrin C et al.; Calreticulin is a 46-kDa Ca(2+)-binding chaperone of the endoplasmic reticulum membranes . The protein binds Ca(2+) with high capacity, affects intracellular Ca(2+) homeostasis, and functions as a lectin-like chaperone . In this study, we describe expression and purification procedures for the isolation of recombinant rabbit calreticulin . The calreticulin was expressed in Pichia pastoris and purified to homogeneity by DEAE-Sepharose and Resource Q FPLC chromatography . The protein was not retained in the endoplasmic reticulum of Pichia pastoris but instead it was secreted into the external media . The purification procedures reported here for recombinant calreticulin yield homogeneous preparations of the protein by SDS-PAGE and mass spectroscopy analysis . Purified calreticulin was identified by its NH(2)-terminal amino acid sequences, by its Ca(2+) binding, and by its reactivity with anti-calreticulin antibodies . The protein contained one disulfide bond between (88)Cys and (120)Cys . CD spectral analysis and Ca(2+)-binding properties of the recombinant protein indicated that it was correctly folded .

Protein Expr Purif, 2000 Nov, 20(2), 179 - 85
Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion; Wang P et al.; Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture . The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography . More than 99% of pro-UK in the culture medium was found in single-chain form . This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion . The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells . However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis . Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK . By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent . It was concluded that glycosylation of pro-UK by yeast P . pastoris interferes with the catalytic site but not secretion of this protein .

Int J Med Microbiol, 2000 Mar, 290(1), 85 - 96
Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus; Reichard U et al.; An aspartic proteinase PEP2 {EC 3.4.23.25} was purified from a cell wall fraction of Aspergillus fumigatus . The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant . A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries . The deduced amino acid sequence of PEP2 consists of 398 amino acids . A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified . The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes . PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger . Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant . Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology . Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.

J Biol Chem, 2001 Jan 26, 276(4), 2678 - 85 Epub 2000 Oct 19.
Site-directed mutagenesis of human ceruloplasmin: . production of a proteolytically stable protein and structure-activity relationships of type 1 sites; Bielli P et al.; A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis . The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains . The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized . The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation . The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame . In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity . This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction . During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene . This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

Chin J Biotechnol, 1999, 15(4), 211 - 7
Expression of transthyretin gene in Pichia pastoris; Ying C et al.; Plasmid pSK-TTR was digested by BamHI and the DNA fragment containing Transthyretin gene was cloned into the BamHI site of vector pHIL-SI . The recombinant plasmid pHIL-SI-TTR was digested by BglII; the larger fragment was transformed into yeast Pichia pastoris . SDS-PAGE analysis showed that the yeast transformant can express and secrete TTR as a fusion protein, TTRF . To purify TTRF,the DEAE-Sepherose F.F . chromatography and Sephacryl S-200 chromatography were used . The in vitro test showed that TTRF could inhibit the growth of human hepatoma cells.

Protein Expr Purif, 2000 Oct, 20(1), 105 - 11
Expression of recombinant galactose oxidase by Pichia pastoris; Whittaker MM et al.; Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product . Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications . Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide . Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase . In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation . Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme . Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield . A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications .

Protein Expr Purif, 2000 Oct, 20(1), 27 - 36
Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme; Dahl SW et al.; A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme . The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da . Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved . The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed . The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein . In all cases, the expressed protein was either undetectable or insoluble . Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity . Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified . Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I . The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis .

Protein Expr Purif, 2000 Oct, 20(1), 1 - 9
Production and characterization of recombinant guamerin, an elastase-specific inhibitor, in the methylotrophic yeast Pichia pastoris; Kim KY et al.; The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography . Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P . pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L . The recombinant guamerin was highly purified (>98%) with a recovery yield of 68% . Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein . The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase . Furthermore, the recombinant guamerin did not produce a humoral immune response in mice .

Tsitologiia, 2000, 42(8), 787 - 93
{Interaction of superhelical DNA with the nonhistone protein HMG1}; Polianichko AM et al.; The interaction between nonhistone chromosomal protein HMG1 and plasmid DNA was studied by optical and hydrodynamical methods . The recombinant protein HMG1 produced by yeast Pichia pastoris strain was used . We have shown that according to the CD spectra local conformational changes in DNA helix occur in the region of DNA-protein interaction . These changes are most significant at r < 3 (w/w) . Both gel-shift assay and ultracentrifugation, as well as CD data, indicate that protein-protein interactions between HMG1 molecules play a major role in the formation of DNA-protein complexes . It is suggested that the protein C-terminus may affect HMG1-DNA binding not only by a direct interaction with DNA helix, but also by protein-protein interactions.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 277 - 86
Yeast secretory expression of insulin precursors; Kjeldsen T; Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli . Here, development of the insulin secretory expression system in S . cerevisiae and its subsequent optimisation is described . Expression of proinsulin in S . cerevisiae does not result in efficient secretion of proinsulin or insulin . However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S . cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g . AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant . The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester . The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S . cerevisiae and Pichia pastories . The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant . However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form . A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield.

Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 428 - 34
Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form; Blum A et al.; 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor . The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification . 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER . In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified . However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed . To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris . The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates . The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver . By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions . Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction . Upon overexpression in the yeast P . pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme . Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase .

Yeast, 2000 Oct, 16(14), 1345 - 50
Construction of fission yeast vectors with a novel selection strategy that allows their use in wild-type fission yeasts; Ingavale SS et al.; Novel vectors that use the Pichia pastoris INO1 gene as a selectable marker and exploit the natural inositol auxotrophy of the fission yeast are described . These plasmids also contained other features desirable in a plasmid cloning vector . These plasmids were evaluated in other species of Schizosaccharomyces and found to replicate autonomously in another variety of S . pombe, S . pombe var . malidevorans . These plasmids can be used for transformation of any wild-type S . pombe strain without the need for selection by induced auxotrophic mutations, or by selection by drug resistance markers, and should greatly assist genetic and molecular manipulations in these yeasts .

J Biomol NMR, 2000 Aug, 17(4), 337 - 47
Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies; Morgan WD et al.; Deuterium isotope labelling is important for NMR studies of large proteins and complexes . Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris . In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P . pastoris grown in deuterated media . The resulting deuteration patterns were analyzed by NMR and mass spectrometry . We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D2O), compared with expression in non-adapted cells . We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D2O medium with protiated methanol as carbon source, or in 95% D2O medium containing deuterated methanol . A high level of uniform Calpha deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in {1H/15N}-correlation experiments was measured . Residual protiation at different positions in various amino acid residues . including the distribution of methyl isotopomers, was also investigated . The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.

Mol Cell Biol, 2000 Oct, 20(20), 7516 - 26
The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import; Collins CS et al.; Peroxisomes are independent organelles found in virtually all eukaryotic cells . Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis . The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins . The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants . We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants . Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p . Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p . Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p . These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation . This hypothesis is supported by the phenotypes of the corresponding mutant strains . As has been shown previously for P . pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 93 - 7
Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation; Veiga A et al.; In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h . Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts . Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts . We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.

Ukr Biokhim Zh, 2000 Mar-Apr, 72(2), 19 - 23
Oversynthesis of riboflavin by yeast Pichia guilliermondii in response to oxidative stress; Protchenko OV et al.; The effect of oxidative stress on riboflavin (vitamin B2) biosynthesis and iron accumulation in flavinogenic yeast P . guilliermondii was investigated . Treatment of P . guilliermondii cells with superoxidgenerating agent methylviologen leads to elevated production of malondialdyhyd (MDA) which reflects the overall cellular oxidation state . Increased iron content in the cells and enhanced productivity of flavinogenesis under these conditions has been shown too . Significant increasing of MDA and riboflavin production by yeast cells under iron deficiency was observed . Riboflavin overproducing P . guilliermondii mutant strains rib80, rib81 and hit, possess high iron transport and synthesize increased quantity of MDA . The role of riboflavin overproduction and activation of iron assimilation in the P . guilliermondii antioxidant defence is discussed.

What Is Cell Biology?, What Is Bioremediation?, What Is Activated Sludge?, What Is Bioengineering?, What Is Bioreactor?, i, Microorganisms, o, Bacterium, s, Microbe, r, Microbes, o, Microbiology, a, Biodegradation, n, Yeasts, r, Gram negative, r, Escherichia coli, s, S. cerevisiae, n, Penicillin, n, Staphylococcus aureus, n, Escherichia coli, e, Streptococci, c, Escherichia coli, n, Proteus, i, Staphylococcus, o, Fermentations, r, Micrococci, e, Bacillus, o, Staphylococcus aureus, a, Haemophilus, o, Escherichia coli, i, Streptococci, n, Culture medium, a, Microorganisms

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005