PCR-based Gene Synthesis, Molecular Cloning, High Level Expression, Purification, and Characterization of Novel Antimicrobial Peptide, Brevinin-2R, in Escherichia Coli

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.