Showing papers on "Pichia pastoris published in 1995"
TL;DR: The Pichia pastoris system has the potential for very high level production of foreign proteins and is now also available for optimizing secretion, such as the generation of clones with progressively increasing vector copy number, expression screening in microtitre plates, and minimizing proteolysis by a number of techniques.
251 citations
TL;DR: It is shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation, and that protein synthesis is required forperoxisome entry into the yeast vacuoles during nutrient adaptation.
Abstract: The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroautophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.
233 citations
TL;DR: The combinatorial immunoglobulin library and phage display technologies are applied to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF).
Abstract: We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.
184 citations
TL;DR: Several new properties of this molecule are reported which show that it is the peroxisomal PTS1 receptor, and the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.
Abstract: The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.
181 citations
TL;DR: The cloning of PER3 and functional characterization of its product Per3p suggest that the vesicles in per3-1 cells are peroxisomal remnants similar to those observed in cells of patients with the peroxISomal disorder Zellweger syndrome, and that the mutant is deficient in PTS1 but not PTS2 import.
114 citations
TL;DR: The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest and is particularly attractive for the secretion of foreign-gene expression cassette.
Abstract: The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growt...
101 citations
TL;DR: The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5‐HT5A receptor and the pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems.
100 citations
TL;DR: The engineered protein (disulfide-stabilized Fv: dsFv) was found to be as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay.
Abstract: Single chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQ kit, Biomira), were constructed in both orientations, i.e. Vh-linker-Vl and Vl-linker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein showed specific anti-idiotype binding activity. Additionally, the molecular graphic modeling has been used to identify a possible site for the introduction of an interchain disulfide bond in the framework region of Fv. These Cys-modifications of the sites were done using a method of PCR-mediated mutagenesis. The engineered protein (disulfide-stabilized Fv: dsFv) was expressed and tested for its binding activity. It was found that dsFv was as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay.
86 citations
TL;DR: Results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM, which is also the largest fragment of human thrombomodulin.
Abstract: Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains. These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain. Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.
75 citations
TL;DR: Because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxISome-inducing environmental conditions, it is concluded that Pas7p is not required forperoxisome proliferation.
Abstract: We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.
68 citations
TL;DR: Western blotting with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other and Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression.
Abstract: Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectin-degrading enzymes are thought to be important for pathogenesis. Antibodies prepared against a pectin-inducible pectate lyase (pectate lyase A [PLA]) produced by a phytopathogenic fungus, Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection. The gene (pelA) and its cDNA were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F. solani f. sp. pisi with the pelA cDNA as the probe. A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced. The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F. solani pisi culture grown in the presence of glucose as the sole carbon source. The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F. solani f. sp. pisi but no significant homology with other pectinolytic enzymes. The first 16 amino acid residues at the N terminus appeared to be a signal peptide. The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa. PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography. Purified PLB showed optimal lyase activity at pH 10.0. A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion. Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other. The 5' flanking regions of both pelA and pelB were translationally fused to the beta-glucuronidase gene and introduced into F. solani f. sp. pisi, and beta-glucuronidase activities of the transformants were measured. Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression. Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F. solani f. sp. pisi infects pea epicotyl.
TL;DR: Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions, and significant clonal variation in the expression of r-ghilanten was found among the His+ transformants.
TL;DR: Alpha-Galactosidase isolated from coffee beans cleaves the terminal alpha-galactose residues from oligosaccharide chains on blood group B red cells, thus generating group O cells, which maintain full erythrocyte integrity and viability in vitro, and demonstrate immune tolerance and a normal life span in vivo.
TL;DR: Important considerations for the use of Pichia pastoris are discussed, including strains for expression, expression plasmids, transformation by integration, and post‐translational modifications.
Abstract: Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.
TL;DR: By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa, and is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
TL;DR: The methylotrophic yeast Pichia pastoris was used as a host system for the production of recombinant APPs, which were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants.
TL;DR: The cDNA encoding human monocyte chemotactic protein 3 was cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris to study antagonism of MCP-3 action in vitro and in disease models of cancer and inflammation.
Abstract: The cDNA encoding human monocyte chemotactic protein 3 (hMCP-3) was cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris. ...
TL;DR: In this paper, a new pectate lyase gene, pelC, was isolated from a genomic library of Fusarium solani f. solani pisi with pelA cDNA as a probe.
TL;DR: This study produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast and engineered a gene coding for a soluble form of CD38 by excision of the region of the genes coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence.
TL;DR: To develop the budding yeast Pichia pastoris as a model system for the study of protein secretion, a secreted acid phosphatase (Pho1p) is characterized from this yeast and it is shown that Pho 1p transits the Pp secretory pathway in less than 5 min.
TL;DR: Functional complementation of a mutant strain, mut2, with a plasmid library of H. polymorpha genomic DNA sequences has identified a gene, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome.
TL;DR: High levels of spinach GO are produced in the methylotrophic yeast Pichia pastoris by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter.
TL;DR: It is demonstrated that N-terminal half-transferrin can easily be expressed in the simple host system, Pichia pastoris, and that the purified protein is capable of reversibly binding iron.
TL;DR: In vitro inactivation/dissociation and subsequent reactivation/re‐assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris and homo‐oligomers are studied.
TL;DR: It is concluded that VLCS is a peroxisomal enzyme in P. pastoris and this organism may serve as an excellent model system to investigate the molecular basis of XALD.
TL;DR: This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins and describes small‐scale preparation of protein extracts.
Abstract: This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins. The protocols listed here for S. cerevisiae are for three widely used types of promoter: galactose-regulated (GAL1, GAL7, GAL10), glucose-repressible (e.g., ADH2), and constitutive glycolytic (e.g., PGK or GAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol-regulated AOX1 promoter and HIS4 selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small-scale S. cerevisiae and P. pastoris cultures and also for high-density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided for S. cerevisiae and yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high-density cultures (e.g., 130 g/liter). Finally, a Support Protocol describes small-scale preparation of protein extracts.
TL;DR: A methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase is reported.
TL;DR: The SUC 2 gene from Saccharomyces cerevisiae coding for the enzyme Invertase was cloned in Hansenula polymorpha under the control of the alcohol-oxidase (AOX) promoter of Pichia pastoris.
Abstract: The SUC 2 gene from Saccharomyces cerevisiae coding for the enzyme Invertase was cloned in Hansenula polymorpha under the control of the alcohol-oxidase (AOX) promoter of Pichia pastoris. The culture conditions for the Invertase production using a fed-batch culture were studied. More than 1,5 × 103 U/ml of Invertase (1 g/L) were found to be secreted to cellular periplasmic space. The scale up to 50 L of the fermentative process was done.
02 Jan 1995
TL;DR: In this article, the effect of stabilization of protein by NaN3 was studied by SERS of flavin adenine dinucleitide released from flavoprotein near the Ag surface.
Abstract: The SERS spectra of alcohol oxidase purified from Pichia Pastoris and Candida Boidinii adsorbed on silver electrode were obtained. The effect of stabilization of protein by NaN3 was studied by SERS of flavin adenine dinucleitide released from flavoprotein near the Ag surface. The dissociation of falvin from flavoprotein in N3-anion from and in neutral form independence on functional state of protein has been supposed. The association of protein oligomers and the peculiarities of quaternary structure are discussed as a reason of different SERS behavior of alcohol oxidase from various different sources.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
TL;DR: The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathePSin E expressed in Pichia pastoris received N-linked high-mannose type glycosylation.
Abstract: The human cathepsin E was purified from the culture supernatant of Pichia pastoris strain transformed with a human cathepsin E expression plasmid.