Isolation, purification, gene cloning and expression of antifungal protein from Bacillus amyloliquefaciens MG-3

Abstract

The antifungal protein MG-3A was isolated from Bacillus amyloliquefaciens MG-3, and was purified and identified. The results showed that antifungal protein MG-3A was likely a serine protease with a molecular weight of ~48 kDa. The serine protease exhibited a broad antifungal spectrum and effectively extended the shelf-life of loquat fruit up to 25 d. The antifungal protein MG-3A showed good stabilities to temperature, pH and protease K. Primers were designed according to the mass spectrum of antifungal protein and the comparison with proteins in the NCBI database. The serine protease gene MG-3A from B. amyloliquefaciens genome was isolated and cloned using PCR. The prokaryotic expression plasmid pET28a-MG-3A was constructed and used to express the antimicrobial protein in vitro. The SDS-PAGE results showed that the recombinant protein expressed in Escherichia coli BL21 (DE3) was highly soluble. Affinity chromatography was used to purify the recombinant protein and its antifungal activity was evaluated.