断裂型内含肽在蛋白生产和生物工程中的应用

蛋白反式剪接是蛋白翻译后修饰的一种特殊机制,这一反应由断裂型内含肽自我催化完成,不需要酶和其他因子参与。与常规顺式的蛋白剪接不同的是,反式剪接是基于断裂型内含肽由N端和C端这两段多肽的高亲和力,精准地构建成一个具有剪接活性的内含肽蛋白质。反式剪接已被发展成新的生物技术应用于生产环化蛋白、构建蛋白定点与定时表达的载体和转基因植物,以及改造cDNA文库技术等。

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

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.