Abstract: 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. Cne PRP8 intein from a eukaryotic genome 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 the 52th site, 68th site,114th site and 146th site amino mutations, and the mutations of its amino acids change the polarity, 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.

Key words: intein, protein splicing, the mechanism of intein splicing