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Showing papers on "Pichia pastoris published in 1996"


Journal ArticleDOI
TL;DR: These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces, indicating that the antigen is produced and developmentally regulated during growth in host tissues.

185 citations


Journal ArticleDOI
TL;DR: It is concluded that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nN OS reductase domain itself.

146 citations


Journal ArticleDOI
TL;DR: Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments.

145 citations


Journal ArticleDOI
TL;DR: Amperometric alcohol biosensors were constructed by co-immobilising commercially available alcohol oxidase with the hydrogen peroxide reducing enzyme, horseradish peroxidase, in a carbon paste matrix and found to yield the best characteristics (sensitivity, selectivity, operational and storage stability, ethanol conversion).

128 citations


Journal ArticleDOI
TL;DR: The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis and cloned the PpPAS2 gene by functional complementation and it is shown that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa.

126 citations


Journal ArticleDOI
TL;DR: Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19 and several differences were found relative to the functional lysosomal α-mannosidase encoded by the 3-kb spleen cDNA.

121 citations


Journal ArticleDOI
TL;DR: Results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.
Abstract: Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.

115 citations


Journal ArticleDOI
TL;DR: The light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria are exploited for studies on the peroxisomal sorting of polypeptides and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is per oxisomal.
Abstract: We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

113 citations


Journal ArticleDOI
TL;DR: The developed method produces more than 80% pure recombinant ovine IFN-tau, obviating the need for further purification for many purposes, and demonstrates the potential of this system for large-scale production of IFN -tau.
Abstract: The early conceptus (embryo and associated membranes) of domestic ruminants signals its presence to the maternal uterus through production of interferon-τ (IFN-τ). Production of IFN-τ ensures conti...

97 citations


Journal ArticleDOI
TL;DR: The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris are rapidly becoming the systems of choice for the expression of recombinant proteins in yeast and the powerful genetic techniques available in Saccharomyces cerevisiae and the fission yeast are still exploited to establish models to study medically important cell processes and screen for pharmacologically active compounds.

93 citations


Journal ArticleDOI
TL;DR: Pichia pastoris produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts, which greatly facilitates future mutational analysis of barley alpha- amylase in order to probe structure/function relationships.

Journal ArticleDOI
TL;DR: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications, which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.
Abstract: BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon) , represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1–specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen. (J ALLERGY CLIN IMMUNOL 1996;98:331-43.)

Journal ArticleDOI
TL;DR: A range of ACE inhibitors were far less potent towards AnCE compared with the humanACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.
Abstract: Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant. Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO). This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE. However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions. Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis. A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.


Journal ArticleDOI
TL;DR: The heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris and the ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino- terminal residues of recombinant proteins.
Abstract: We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.

Journal Article
TL;DR: The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per 6p is the putative yeast homolog of PAF -1.
Abstract: We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif, Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with carl, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina, In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and carl, Like PAF-I, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent, Instead, peroxisomal remnants are observed, In addition, peroxisomal matrix proteins are synthesized but located in the cytosol, The similarities between Per6p and PAL-I in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF 1.

Journal ArticleDOI
TL;DR: The human μ‐opioid receptor cDNA from which the 32 amino‐terminal codons were substituted by the Saccharomyces cerevisiae α‐mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase‐1 gene.

Journal ArticleDOI
TL;DR: A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leech Hirudo medicinalis, was cloned and expressed in the methylotrophic yeast Pichia pastoris, offering an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.

Journal ArticleDOI
TL;DR: Per6p as discussed by the authors is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif that is essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris.
Abstract: We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.

Journal ArticleDOI
TL;DR: This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart.

Journal ArticleDOI
TL;DR: A method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies is developed.

Patent
08 Nov 1996
TL;DR: In this paper, a methanol-inducible promoter from an alcohol oxidase gene, such as Pichia pastoris AOX1, was used to regulate GAD65 expression.
Abstract: Methylotrophic yeast are used for high-level expression of GAD65 that makes the production of GAD65 feasible on an industrial scale. A methanol-inducible promoter from, for example, an alcohol oxidase gene, such as Pichia pastoris AOX1, can be used to regulate GAD65 expression. The recombinant GAD65 has high specific activity and retains antigenic characteristics of the native molecule that are essential to immunological assays and therapeutic protocols.

Patent
04 Jun 1996
TL;DR: A method of producing hexose oxidase by recombinant DNA technology, recombinant Hexose Oxide oxidase and the use of such enzyme, in particular in the manufacturing of food products such as doughs and dairy products, animal feed, pharmaceuticals, cosmetics, dental care products, and manufacturing of lactones is discussed in this article.
Abstract: A method of producing hexose oxidase by recombinant DNA technology, recombinant hexose oxidase and the use of such enzyme, in particular in the manufacturing of food products such as doughs and dairy products, animal feed, pharmaceuticals, cosmetics, dental care products and in the manufacturing of lactones. Suitable sources of DNA coding for the enzyme are marine algal species including Chondrus crispus, Iridophycus flaccidum and Euthora cristata . In useful embodiments, the recombinant hexose oxidase is produced by Pichia pastoris, Saccharomyces cerevisiae or E. coli.

Journal ArticleDOI
01 May 1996-Yeast
TL;DR: Human single‐chain urokinase‐type plasminogen activator without an N‐glycosylation site (scu‐PA‐Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR).
Abstract: Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0.47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q302 were attempted. Addition of Triton X-100, L-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q302 and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q302 closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.

Journal ArticleDOI
30 Sep 1996-Yeast
TL;DR: The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextanase cDNA as a probe and comparison of the gene and cDNA sequences revealed that theDEX gene does not contain introns.
Abstract: The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicilliurri niitziolirteutii by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. tniniolutwm dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobucter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichiu pusroris by using the SUC2 gene signal sequence from Sncchnroinyces rerevisiae under control of the alcohol oxidase-1 (AOXI) promoter. Over 3.2 gA of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.

Journal ArticleDOI
TL;DR: Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics.
Abstract: N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation. The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39-kDa and 59-kDa peptides, respectively). The absorption properties and photochromicity upon red/far-red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N-termini, but C-terminal positions identical to those of the corresponding tryptic fragments (45-kDa and 65-kDa peptides). All recombinant 65-kDa and 59-kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the Pr and the Pfr forms. The smaller chromopeptides (45-kDa and 39-kDa) behaved differently depending on the expression system employed. E. coli-derived peptides exhibited a phytochrome-like difference spectrum only when the intact N-terminus was present (45-kDa product). The recombinant 39-kDa pep-tide from E. coli was incapable of chromophore binding whereas the identical peptide sequence expressed by P pastoris formed a chromoprotein with phycocyanobilin. This recombinant phytochrome fragment exhibited a difference spectrum (Pr–Ppr) with an even larger Pfr absorption band than the comparable tryptic 39-kDa fragment. Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics. Evidently, trypsin digestion down to the 39-kDa fragment affects protein conformation also in terms of Pfr conservation.

Journal ArticleDOI
01 Jan 1996-Gene
TL;DR: It is shown that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp, suggesting that transcription both initiates and terminates at the same sites in both yeast species.

Journal ArticleDOI
TL;DR: A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris and overexpressed a modified beta- cryptogein in a secreted form.

Journal ArticleDOI
TL;DR: Investigation of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins, supporting the conclusion that this species possesses a phy tochromobilin prosthetic group.
Abstract: Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

Journal ArticleDOI
TL;DR: The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins, and the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory.
Abstract: The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.