低温菌启动子分析及异源蛋白高效表达

The Analysis of Promoters and the Effective Expression of Exogenous Proteins in a Cold-adapted Bacterium

在已构建的能在低温菌和E.coli中正常复制的启动子探针质粒的基础上,筛选到了强启动子,通过RT-PCR评估了启动子活性,并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白表达质粒,使一个热不稳定α-淀粉酶在低温下(7℃)得到了高效表达,表达量达胞外总蛋白的35%。显示出该表达系统具有高效表达热不稳定蛋白质的潜在应用价值。

Based on the constructed promoter probe vectors that could replicate both in E. coli and in a cold-adapted bacterium, several candidate promoters were isolated and their activities were evaluated by RT-PCR. The transcription initiation sites and core sequence of promoters were determined by primer extension analysis. A low-temperature expression vector was constructed by using the strongest promoter and a thermolabile α-umylase gene was successfully overproduced under control of this promoter at low temperature （7℃）, while the secreted α-umylase amounted up to 35% of the total extracellular proteins. The expression system is expected to be useful for the production of thermolabile exogenous proteins at low temperatures.