单启动子双拷贝CPC酰化酶基因在毕赤酵母中的表达

为了实现外源基因在毕赤酵母中的高效表达，以口蹄疫病毒的2A片段连接2个头孢菌素C (CPC)酰化酶基因，形成双顺反子结构的质粒，整合到毕赤酵母的基因组中，并以筛选标记和聚合酶链式反应进行验证，最终得到双拷贝的阳性重组子. 通过摇瓶发酵培养，双拷贝转化菌株的最高酶活为2810 U/L，而在相同菌体浓度下，双拷贝转化菌株的CPC酰化酶酶活为单拷贝转化菌株的1.6倍. 利用结构短小和高剪切效率的2A多肽，不但可达到单启动子调控多份基因的目标，也为真核表达系统中以基因的拷贝数增强外源蛋白的表达提供了简便且高效的思路.

Expression of Cephalosporin C Acylase by Bicistronic Vector in Pichia pastoris

In order to effectively express heterologous proteins in Pichia pastoris, the plasmid with bicistronic structure that connect two cephalosporin C (CPC) acylase genes with foot-and-mouth disease virus (FMDV) 2A sequence was constructed. After two-copy recombinant plasmid was integrated into the Pichia pastoris genome, positive recombinants were identified with selectable markers and PCR amplification. The optimal CPC acylase productivity of two-copy transformant reached 2810 U/L in shaking flask fermentation, the CPC acylase activity of two-copy transformant was 1.6 times of one-copy one at the same condition of cell concentrate. Thus, the 2A peptide with short structure and effective self-cleavage reaction not only achieved the goal of regulating multiple genes by a single promoter, but also provided a simple and effective thinking for improving expression of heterologous proteins by gene copy number.