Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression
Research article published in Sheng Wu Gong Cheng Xue Bao (2001)
Abstract
Pichia pastoris has become an increasingly popular host for heterologous protein production. However, there is neither a high-efficient transformation method nor a fast colony-PCR assay for the yeast yet. In this paper, we report a transformation procedure by electroporation, which reaches the value of up to 2800 transformants/microgram DNA. By using a cold and heat treatment and a modified PCR buffer, we established a simple and reliable colony-PCR protocol to detect recombinant P. pastoris clones, which is comparable to the conventional assay for E. coli colonies. With these two novel techniques, we have successfully achieved the expression of hirudin, an antithrombin agent, in Pichia pastoris. The secreted hirudin maintains a biological activity of 82 antithrombian units per milliliter supernatant from the media.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
Transformation procedure by electroporation reaching 2800 transformants/microgram DNA for Pichia pastoris; expression of hirudin in P.
Why This Matters for Hirudotherapy
This study reports improved methods for genetic manipulation of Pichia pastoris—an electroporation transformation protocol (up to 2800 transformants/microgram DNA) and a colony-PCR assay—and applies them to express hirudin, an antithrombin agent, secreted at 82 antithrombin units per milliliter of culture supernatant. This is directly relevant to ASH because hirudin is the canonical anticoagulant of medicinal leech saliva and a defining molecule of the leech secretome that underpins leech therapy's anticoagulant effects; recombinant production platforms are important for enabling reliable supplies of hirudin and hirudin-based therapeutics for research and clinical use. A caveat: this is a bioprocess/expression study, not a clinical or in-vivo efficacy study, and the reported activity reflects expression yield in culture supernatant rather than purified product performance or comparison with native leech-derived hirudin.
Citation
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