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

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 

机构地区：[1]Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China [2]New England Biolabs, Inc., Beverly, Massachusetts 01915, USA

出　　处：《Chinese Science Bulletin》2006年第13期1652-1654,共3页

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 pro- teins 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-termi- nal 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 pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.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. coil ER2799. The G2-EPSPS gene was then divided into N-termi- nal 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.

关 键 词：断裂基因 蛋白质再生 转基因 EPSPS 

分 类 号：Q78[生物学—分子生物学]