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.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: The importance of codon optimization and clone selection

Abstract: In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris

Abstract: Interleukin-1 receptor antagonist (IL1ra) is known to treat a number of diseases such as rheumatoid arthritis and type 2 diabetes. However, the biological half-life of IL1ra is very short due to its rapid renal clearance. Our present study aimed to increase the biological half-life of IL1ra through fusion with human serum albumin (HSA), and then augmented expression of the IL1ra and HSA fusion protein (IH) in Pichia pastoris strain by increasing IH gene copy number or was co-expressed with chaperone. By comparing clones containing varying copy numbers of IH fusion gene, it was observed that higher levels of secretory IH fusion protein was produced in strain with higher IH gene copy number. In addition, IH protein yield was further improved after being co-expressed with protein disulfide isomerase (PDI). Conversely, it was significantly decreased (i.e., secretory IH in the culture medium) by co-expression of immunoglobulin binding protein. We have also discussed whether the multi-copy strain and co-expressed of PDI could enhance the levels of other secretory albumin fusion protein (e.g., HSA and human growth hormone fusion protein). Interestingly, the level of this fusion protein was apparently also increased by these approaches. In conclusion, our results have demonstrated that increasing copy number and co-expression of PDI may raise yield of albumin fusion protein in P. pastoris, which might probably contribute to the industry for the development of proteinous drugs.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel

Abstract: We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.