摘要
根据毕赤酵母（Pichia pastoris）密码子选择偏好对来源于芽孢杆菌（Bacillus circulans）的乳糖酶基因lacO进行了分子改造。改造后的基因lacM按正确的阅读框架插入到毕赤酵母表达载体pPIC9上，构建得到重组毕赤酵母表达质粒。PCR检测、SDS—PAGE电泳分析和酶活力测定的结果表明，lacM基因已重组到酵母染色体上，并能正常分泌表达。与来改造的基因lacO的毕赤酵母重组子相比，酶蛋白表达量明显增加，约为改造前的3倍以上。
The lacO gene from Bacillus circulansatut encoding lactose was modified to the lacM gene without changing its amino acid sequence, according to the bias in codon choice of yeast. The lacM gene was cloned into vector of pPIC9 correctly and constituted the recombinant vector of pPIC9-LacM. A high-expression strain was constructed by transforming pPIC9-LacM into Pichia pastoris GSllS. The SDS-PAGE analysis and enzyme activity assay revealed that the recombinant lactose gene was expressed highly in Pichia pastoris. The expressed product can be secreted effectually and had the normal bioactivity. The enzyme activity of recombinant protein LACM was more than 3 folds of that with unmodified lacO gene in recombinant Pichia pastoris.
出处
《高技术通讯》
CAS
CSCD
北大核心
2006年第1期55-60,共6页
Chinese High Technology Letters
基金
863计划（2004AA214041）资助项目.
