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 coll