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.

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

Abstract: 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.

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity.

Abstract: Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 ± 32 nmol min-1 mg-1. The ATPase activity ...

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris.

Abstract: The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.

Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris.

Abstract: The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx, 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit alpha 1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar K(m) for mannose (280-350 mM), methyl-alpha-mannoside (60-90 mM) and GDP-mannose (50-90 microM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-alpha-mannoside, alpha-1,2-mannobiose and methyl-alpha-1,2-mannobiose, as well as Man15-30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize alpha-1,6-mannobiose, alpha-1,6-mannotriose, alpha-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9-10GlcNAc. These results indicate that Kre2p/ Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.