Expression, Purification and Characterization of a Novel Antimicrobial Peptide: Gloverin A2 from Bombyx mori

Abstract

Worldwide, antibacterial resistance has increased dramatically in the past years. Antimicrobial peptides are a group of immune-related peptides/proteins that protect the host from microbial infections, and were identified broad activity against pathogenic microorganism and not easy to generate insecticide resistance. It appears that antimicrobial peptides possess great potential for developing into a new ideal antimicrobial drug. Bombyx mori Gloverin A2 (BMGlvA2) was an antimicrobial peptide of B. mori, however, directly extracted and purified BMGlvA2 was difficult and uneconomic. In this study, BMGlvA2 gene was successfully cloned from B. mori and heterologous expression with vector pET28a (+) by E. coli Rosetta (DE3). The selected E. coli Rosetta (DE3)-BMGlvA2 strain yielded 241 µg/ml recombinant BMGlvA2 (rBMGlvA2) protein after 9 h inducted by 1.0 mM IPTG (isopropyl-β-d-thiogalactoside) at 28 °C. rBMGlvA2 protein was identified by western blot and MALDI-TOF. MALDI-TOF analysis indicated that its amino acid sequence is consistent with naturally secreted proteins by B. mori (sequence coverage 78.71%). Importantly, inhibition zone assay shown that rBMGlvA2 efficiently inhibited the growth of several Gram-negative bacteria, including E. coli DH5α, E. coli Rosetta, E. coli K88, S. typhimurium and several Gram-positive bacteria, including S. aureus and B. subtilis. Furthermore, The MIC of rBMGlvA2 showed antimicrobial activity against E. coli DH5α. Meaningfully, rBMGlvA2 did not display hemolytic activity against pig red blood cells. In conclusions, the antimicrobial activity and hemolytic of rBMGlvA2 suggested that it could be one of potential alternatives to conventional antibiotics against Gram-negative and Gram-positive bacteria.