The Protein Splicing Of Cne PRP8 Intein

Inteins have been defined as protein sequences embedded in-frame within a precursor protein equence and excised during a maturation process termed protein splicing.Intein-mediated protein splicing offers many promises in protein engineering.However the mechanisms of protein splicing is still unclear,which greatly limits the general application of protein splicing in protein engineering.This project studies Cne PRP8 intein that was from a eukaryotic genome and had been improved through directed evolution consisting of random mutagenesis followed with functional selection.This improved Cne PRP8 intein will be subjected to site-specific mutations of amino acid residues at critical locations,followed with restoration mutation,and mutational studies of extein amino acid residues at the splice junctions,in order to discover new mechanisms of the protein splicing.The results show that the splicing of eukaryotic protein intein is still related to its one site and its positive one site,but for the intein of Cne PRP8 protein,its splicing efficiency is related to its 52 site,68 site,114 site and 146 site amino acid mutations,and the mutations of its amino acids changed the spatial conformation of the active center,thus affecting the splicing of protein introns.This study not only consolidates the mechanism of protein intron splicing,but also reveals the evolutionary pathway of protein introns in eukaryotes.But still need further exploration and research.