Construction And Optimization Of Split Functional Inteins In Front Of A Thr Residue

Intein is a protein-intervening sequence that catalyzes a protein-splicing reaction in which the intein sequence is precisely excised and its flanking sequences (N-exteins and C-exteins) join with a peptide bond to produce the mature host protein (spliced protein). Inteins can be categorized into three types:bi-functional inteins, containing a splicing domain interrupted by a homing endonuclease domain; mini-inteins, containing only a splicing domain; and split-inteins, consisting of separately encoded N-intein and C-intein that could reassemble through structural complementation to catalyze trans-splicing. The mechanism of protein splicing typically has four steps:N-O or N-S acyl rearrangement; transesterification reaction; cyclization of the conserved intein C-terminal Asn; O-N or S-N acyl rearrangement. The first C-extein(+1) residue is conserved serine, cysteine or threonine (Ser, Cys or Thr), and it is very important to protein self-splicing, attacking a peptide bond at the N-terminal splice junction, alleviating the need to form the initial (thio)ester at the N-terminal splice junction, thus promoting the splicing process. The intein sequence plus the first C-extein residue generally contain all the necessary structural information for protein splicing. Most of the first C-extein residue was found to be Cys or Ser, only 17% of known inteins'first C-extein residue were Thr, while Thr was found to be more common in target proteins than especially Cys. But only C+1 and S+1 inteins can be used in research, and they just excute their function before Cys and Ser, though losing its activity before Thr. With no doubt this restrain the application, we need more useful split intein. Choosing the best high splice efficiency mini-intein from them to constructure into split intein. The self-splicing activities of these artificially inteins were assessed in a protein splicing test system in vivo and vitro. At the same time, Tempreture of inducing of splicing have been optimized. we found Snf-SO have high splice ability; Research the splice is influencely by conservative amino acid, shows T+1 split intein with splicing activity, it not only splice before Thr, but also can splice no matter+1 site is Cys or Ser; When+1 site is Ser, its splicing activity is close to Thr is in+1 site.Snf-m derived from Trichodesmium erythraeum Ter Snf2 inteins were selected to improve with directed evolution strategy. Error-prone PCR is used in directed evolution of Snf-m. A mutant library was constructed, then the mutants were screened in a kanamycin resistance-dependent system. The coding sequences of Snf-m were cloned in kanamycin resistant gene, which coding for phosphortransferase. The resistance of kanamycin is destroyed by the changes of structure of phosphortransferase, but resumed if protein splice occurred in phosphortransferase. Three rounds of error-prone PCR were done. The protein splicing activity of all the mutants was identified using Western blotting. No positive mutant was obtained in the third round and four round.This study will provide more abundant materials in the application of inteins. The analysis of intein mutants will provide more information about the relationship between the splicing mechanism and structure, and more clews in the origin and evolution of this self-splicing element.