Directed Evolution, Cleavage, Segregate Expression Of Inteins In Vivo And Splicing In Vitro

Intein is a protein-intervening sequence,and its flanking sequences are called extein.By four nucleophilic displacement reactions intein catalyzes exteins to ligate with a peptide bond to produce the mature host protein,with itself precisely excised from precursor protein in the post-translational maturation process.This intein mediated unique autocatalytic biochemical reaction provided us a new method for protein synthesis and modification.There is a wide perspective for further applications of intein-based protein engineering.However,there are a lot of problems unsettled by now in this field,such as the low efficiency of splicing in heterologous hosts,the low yield of intein mediated purification system and the impracticability of artificial control on the process of splicing.In order to solve the problems above,several changes were made on inteins as follows:By combining the splicing activity of intein with kanamycin resistance,a whole set of kanamycin resistance-based screening system of intein was established in heterologous hosts.Ter ThyX and Ssp DnaX mini intein were used to analyze the feasibility of this system.The results that showed on the plate containing kanamycin exhibited high similarity to western blot,which indicated that the screening system for active intein was established successfully.Based on directed evolution,a mutation library more than 10~6 colonies was set up by error prone PCR.Four mutational inteins with high splicing efficiency were screened on the plate containing kanamycin.The result of western blot showed that the reformed Ssp DnaX mini intein can splice with more than 90%activity in E.coli, which tackled the problem of the low efficiency of splicing in heterologous hosts.S1 and S11 split intein of Rma DnaB and Ssp GyrB were used to construct N-or C-cleavage element by site-specific mutagenesis on N-or C-terminal.With the conservative amino acids related to splicing changed,the new inteins lose their activities of splicing and only remain the function of cleavage on the terminal.The efficiency of cleavage was analyzed to extract a high activity element,which can be used to do further research on the mechanism of splicing and establish a high yield of intein mediated purification system.A fusion protein(NC protein) contained two short fragments of polypeptide on N- and C-terminal was co-expressed in E.coli.Another middle fragment of intein(IM protein) was expressed,too.The NC and IM protein were purified respectively in vitro.And then the two fragments were spliced in vitro at the temperature of 16℃,25℃,37℃.The reaction can promote the formation of terminal dimerization so that we can reason out the answer to the specific binding between IN and IC.In case of the unknown higher structure of intein,intein with high activity of splicing was screened to fulfil the evolution of function.Spatial isolation was used to study the reaction between fragments of intein,and to lay the foundation of research on the controllability of splicing.With the development and progress on the study of intein,it will be a powerful tool in different fields of protein engineering.