Site-directed mutagenesis of microbial transglutaminase gene by overlap extension PCR

Objective To investigate the site-directed mutagenesis of microbial transglutaminase(MTG)by overlap extenision PCR.Methods Primers were designed by using DNAMAN5.0 software based on the MTG sequence of Streptomyces sp.H197 in GenBank and the principle of overlap extension PCR-based site-directed mutagenesis,with which MTG gene was amplified by PCR and used as a template for site-directed mutagenesis by overlap extension PCR. PCR product was recovered and inserted into vector pMD-19T,and the constructed recombinant plasmid pMD-19T-MTG was transformed to E.coli DH5α for identification by PCR,restriction analysis and sequencing.Results A specific gene fragment containing restriction and mutation sites was observed on electrophoretic profile of overlap extension PCR product.PCR and restriction analysis proved that recombinant plasmid pMD19-T-MTG was constructed correctly.Sequencing result showed mutation at only one site(A→G at site 773)as compared with the sequence of wild type MTG,which was consistent with the expected result.Conclusion The site-directed mutagenesis of MTG gene was successfully achieved by overlap extension PCR,and the positive clones contained mutation site,which laid a foundation of study on function of mutant.