Improved Protein Splicing By Directed Evolution Of Inteins In Heterogenous Host

Intein is an internal protein sequence in host protein precursor.It can autocatalytically self-excise and concomitantly splice together the flanking polypeptides with a peptide bond.This process is termed as protein splicing.This intein-mediated protein ligation approach opens up novel routes in protein engineering.But there are many restricts in its application,such as low amount of native inteins have been found and their splicing activity decrease dramatically or even totally lose when assayed in heterogenous proteins.It is hard to improve the splicing activity in a rational design way,which based on clear understanding of the relationship between the protein splicing mechanism and intein structure.In this study,inteins with low splicing activity were evolved with directed evolution strategy.The aim is to obtain more applicable inteins with high protein splicing activity.Four native inteins Ter DnaB-1,Ter DnaE-3,Ter RIR1-3 from Trichodesmium erythraeum and Cne-AD PRP8 from Cryptococcus neoformans serotype AD were reconstructed to artificially mini-inteins and split inteins.The self-splicing activities of these artificially inteins were assessed in a protein splicing test system in Escherichia coli,which constructed with meltose binding protein and thioredoxin.The results indicated that splicing occurred efficiently of Ter DnaE-3 and Cne-AD PRP8,while failed of Ter DnaB-1 and Ter RIR1-3. Combining with protein splicing study of other inteins in our lab,two artificially mini-inteins,TEI-m and TE2-m derived from Trichodesmium erythraeum DnaE-1 and DnaE-2 inteins were selected to evolve.Error-prone PCR was applied in directed evolution of TE1-m and TE2-m.A mutant library was constructed,and the mutants were screened in a kanamycin resistance-dependent system.The coding sequences of TE1-m and TE2-m were cloned into kanamycin resistant gene,which coding for phosphotransferase.The resistance of kanamycin is destroyed by the changes of structure of phosphotransferase,but resumed if protein splice occurred in phosphotransferase.Three rounds of error-prone PCR were done.The first round of mutants was screened on medium containing 5μg/ml kanamycin.20 positive mutants were obtained.The second round of mutants was screened on medium containing 10μg/ml kanamycin,and 11 positive mutants were obtained.No positive mutant was obtained in the third round of screening on medium containing 20μg/ml kanamycin.The protein splicing activity of all the mutants was identified with Western blotting.One positive mutant pK⊿TE2-⊿G was obtained after two rounds of error-prone PCR,which showed more efficient splicing.There are three amino acid mutations in this mutant,which are I52F,P100S and A-1G,respectively. I52F and P100S are located in TE2-m,while A-1G located outside the intein, which is the first N-extein residue flanking the N terminal of intein.Further study indicated that the three mutations resulted in protein splicing together.This study indicates that directed evolution strategy is efficient in improving the protein splicing activity.It will provide more abundant materials in the application of inteins.The analysis of intein mutants will provide more information about the relationship between the intein structure and protein splicing mechanism, and more clews in the origin and evolution of this self-splicing element.