Cloning, expression and characterization of antimicrobial porcine β defensin 1 in Escherichia coli

Abstract: Porcine β defensin 1 (pBD1) is a cationic antimicrobial peptide with three pairs of disulfide bonds. When expressed in insect cells, two polypeptides of different length (pBD138 and pBD142) accumulated, which differed by N-terminal truncation. However, only pBD142 was found in pigs. pBD142 had stronger antimicrobial activity than pBD138, and thus could be a good candidate as a bactericidal agent for pigs. In this study, pBD142 gene, obtained by RT-PCR using the tongue total RNA as a template, was cloned into pET30a expression vector and transformed into Escherichia coli BL21 (DE3) plysS. The recombinant pBD142 was expressed after induction by IPTG and purified by His tag affinity column with 90% purity. The recombinant pBD142 exhibited antimicrobial activity against both Gram-positive Staphylococcus aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentrations (MICs) of recombinant pBD142 against tested bacteria were 100μg/mL for E. coli and 80μg/mL for S. aureus. In addition, pBD142 showed low hemolytic activity and high thermal stability. These properties are relevant for the biotechnological applications of the peptide. [Copyright &y& Elsevier]