%0 Journal Article
%T Technical Improvements in Genetic Manipulation of Pichia pastoris and Their Application in Hirudin Expression<br>毕赤酵母基因操作技术的改进及其在水蛭素表达中的应用
%A CAI Chuan
%A |Qi
%A FANG Rong
%A |Xiang
%A <br>蔡传奇
%A 方荣祥
%J 生物工程学报
%D 2001
%I 
%X 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.
%K Pichia pastoris\%
%K transformation by electroporation
%K colony\|PCR
%K hirudin<br>毕赤酵母Pichia
%K pastoris
%K 电穿孔转化，
%K 菌落PCR，
%K 水蛭素
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=1F6B592D879D7A50&yid=14E7EF987E4155E6&vid=BCA2697F357F2001&iid=0B39A22176CE99FB&sid=7CE3F1F20DE6B307&eid=1B97AE5098AEB49C&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=14&reference_num=11