Analysis Of Insertion Mutation Sites In Cytidine Deaminase By Transposon

In protein engineering, different synthetic protein switches can be constructed by splitting or inserting new domains at specific sites of the protein, but how to find the specific insertion sites in the protein limits the construction of these synthetic protein switches. Therefore, it is particularly important to find a suitable method to screen all possible insertion sites in the target protein.In our study, we constructed an in vitro transposition reaction system based on transposon, after optimization, we could quickly and efficiently construct transposon random insertion library which could meet the requirements of diversity and homogeneity. Next, we chose activation induced deaminase (AID) protein as our target, AID protein could convert C to T at the single base level, a lot of single base gene editing tools were successfully constructed based on this property of the AID protein and its family members, thus understanding the potent insertion sites within the AID protein is of important significance to the subsequent use of the AID protein.To this end, we constructed an library with transposon randomly inserted into the AID protein, through further digestion and ligation, two different types of fragments(GGGGS)2 and (EAAAK)2 were separately inserted into the AID protein. Finally, two tolerance regions : loop7 and C terminal were screened by rifampicin mutagenesis assay method, by combining error prone PCR with cyclic enrichment screening, we successfully screened a series of error prone PCR mutants with insertion mutation,these mutants could show higher activity than wild type AID protein in different degree, this provided an experimental basis for the further use of the AID protein.