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 

作者机构：Biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing 100081 China New England Biolabs Inc. Beverly Massachusetts 01915 USA 

出 版 物：《Chinese Science Bulletin》 (Chin. Sci. Bull.)

年 卷 期：2006年第51卷第13期

页      面：1652-1654页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：National High Technology Project “863”, (2004AA-214170) National Project “973”, (001CB108904) 

主　　题：断裂基因 蛋白质再生 转基因 EPSPS 

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 *** 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.