[Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression]

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.