American Society of Hirudotherapy

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)

Last Updated: June 18, 2026Reviewed by: ASH Editorial Board
Research article — evidence reviewArticle reference
Evidence: Research reportSalivary PharmacologyDrug DevelopmentCai CQ et al. · 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.

Publication typeEnglish AbstractJournal Article
Indexed MeSH termsElectroporationHirudinsPichiaPolymerase Chain ReactionTransformation, Genetic

Summary

An improved electroporation procedure yielding up to 2800 transformants per microgram DNA, together with a rapid colony-PCR assay, enabled expression of antithrombin hirudin in Pichia pastoris at 82 antithrombin units per milliliter.

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 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

Reference (Vancouver style)

Cai CQ, Fang RX. Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression. Sheng Wu Gong Cheng Xue Bao. 2001;17(2):155-160. PMID: 11411222

Bibliographic details (authors, volume, pages, DOI) are taken from the PubMed record.

PubMed

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