Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

文献类型： 外文期刊

第一作者： Dun, Baoqing

作者： Dun, Baoqing;Lu, Wei;Zhang, Wei;Ping, Shuzhen;Wang, Xujing;Chen, Ming;Xu, Yuquan;Jin, Dan;Wang, Jin;Zhao, Zhonglin;Liang, Aimin;Hou, Songna;Xu, Ming-Qun;Lin, Min

作者机构：

关键词： splitting gene;intein;protein reconstitution;transgene

期刊名称：CHINESE SCIENCE BULLETIN （ 影响因子：1.649； 五年影响因子：1.738 ）

ISSN： 1001-6538

年卷期： 2006 年 51 卷 13 期

页码：

收录情况： SCI

摘要： A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E coli ER2799. The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPSN2951N and pKEPSc2961c. Co-transformation of plasmids, pMEPSN2951N and pKEPSc2961c, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

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