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.
Zusammenfassung
Transformation procedure by electroporation reaching 2800 transformants/microgram DNA for Pichia pastoris; expression of hirudin in P.
Warum dies für die Hirudotherapie relevant ist
This article reports improved genetic manipulation techniques for Pichia pastoris — specifically an electroporation transformation method reaching up to 2800 transformants/microgram DNA and a colony-PCR protocol for detecting recombinant clones — and applies them to express hirudin, described as an antithrombin agent. Secreted hirudin showed biological activity of 82 antithrombin units per milliliter of supernatant. The abstract describes hirudin only as an antithrombin agent and makes no mention of leeches, the leech secretome, or hirudotherapy. There is no defensible connection to ASH's domain based on this abstract. This is a methodological study in yeast with no clinical or in-vivo data.
Zitation
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