Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.

High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture

Abstract: Background Aeromonas hydrophila is a serious pathogen and can cause hemorrhagic septicemia in fish. To control this disease, antibiotics and chemicals are widely used which can consequently result in "superbugs" and chemical accumulation in the food chain. Though vaccine against A. hydrophila is available, its use is limited due to multiple serotypes of this pathogen and problems of safety and efficacy. Another problem with vaccination is the ability to apply it to small fish especially in high numbers. In this study, we tried a new way to attenuate the A. hydrophila infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in Pichia pastoris.

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Abstract: Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.

Mutations in Either Nucleotide-Binding Site of P-glycoprotein (Mdr3) Prevent Vanadate Trapping of Nucleotide at Both Sites †

Abstract: Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Abstract: Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

Abstract: We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.