Improved Cne PRP8 By Directed Evolution

Inteins have been defined as protein sequences embedded in-frame within a precursor protein sequence and excised during a maturation process termed protein splicing. Protein splicing is a post-translational event involving precise excision of the intein sequence and concomitant ligation of the flanking sequences by a normal peptide bond. Inteins play important roles in the biological research and biotechnology now, such as creating cyclical proteins,segmentally labeled. However, mostly the splicing efficiency of inteins is not high, and sometimes they are inactive when invading a different host protein. Based on the limited structural information, this method of directed evolution can sometimes result in great improvement of intein splicing.This technique can generate a large number of mutants in a very short period of time, and it doesn't depend on the structure information to gain proteins of certain properties directedly.In the study, the coding sequence of Cne PRP8 was firstly inserted the gene for the KanR,and then subjected to error-prone PCR and Kanr screening, An improved Cne PRP8, namely CPE, was gotten, which have more general and have higher splicing efficiency. And the split-CPE behaved well splicing, which indicates the CPE will have large prospects in practice. In additions, several amino acids proved to be important to the splicing of CPE,and benefit to further researching the relationship between its structure and function.