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.

Sequential deletion of Pichia pastoris genes by a self-excisable cassette

Abstract: A rapid and convenient method is presented for unmarked gene deletions in Pichia pastoris. Cre/mutated lox system, Zeocin(®) (Invitrogen) resistance marker and homologous arms were spliced together by fusion PCR to generate the gene disruption cassettes (homologous region-lox71-Cre-ZeoR-lox66-homologous region), which could be integrated into the P. pastoris genome via homologous recombination. After transferring double-cross-over recombinants to methanol induction medium, transient expression of Cre recombinase caused the recombination of lox71-Cre-ZeoR-lox66 fragment into a double-mutant lox72 site, thus excising the Cre-ZeoR cassette from the P. pastoris genome. As the double-mutant lox72 site displays strongly reduced binding affinity for Cre recombinase, this method could be used sequentially to disrupt P. pastoris genes without introducing selectable markers. The effectiveness of this strategy was verified by introducing both single and double gene deletions into the P. pastoris genome.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

Abstract: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics The methylotropic P pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers These recombinant antibody derivatives were efficiently expressed in both mammalian and P pastoris cells Stable production in NS0 cells produced 1305 mg pure bibody and 27 mg pure tribody per litre This high yield is achieved as a result of the high overall purification efficiency of 77% Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts Additionally, the yields of 368 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E coli Bi- and tribody molecules are efficiently produced in P pastoris Furthermore, the yeast produced molecules retain the same specific affinity for their antigen These results establish the value of P pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives

High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10

Abstract: The yeast Pichia pastoris (P. pastoris) has become a popular `cell factory' for producing heterologous proteins, but production widely varies among proteins. Cultivation temperature is frequently reported to significantly affect protein production; however, the underlying mechanisms of this effect remain unclear. A P. pastoris strain expressing recombinant human interleukin-10 (rhIL-10) under the control of the AOX1 promoter was used as the model in this study. This system shows high-yield rhIL-10 production with prolonged methanol-induction times when cultured at 20°C but low-yield rhIL-10 production and higher cell death rates when cultured at 30°C. Further investigation showed that G3-pro-rhIL10, an immature form of rhIL-10 that contains the glycosylation-modified signal peptide, remained in the ER for a prolonged period at 30°C. The retention resulted in higher ER stress levels that were accompanied by increased ROS production, Ca2+ leakage, ER-containing autophagosomes, shortened cortical ER length and compromised induction of the unfolded protein response (UPR). In contrast, G3-pro-rhIL10 was quickly processed and eliminated from the ER at 20°C, resulting in a lower level of ER stress and improved rhIL-10 production. High-temperature cultivation of an rhIL-10 expression strain leads to prolonged retention of immature G3-pro-rhIL10 in ER, causing higher ER stress levels and thus greater yeast cell death rates and lower production of rhIL-10.