Antimicrobial characterization of site-directed mutagenesis of porcine beta defensin 2.

Porcine ß defensin 2 (pBD2) is a small, cationic and amphiphilic antimicrobial peptide. It has broad antimicrobial activities against bacteria and plays an important role in host defense. In order to enhance its antimicrobial activity and better understand the effect of positively charged residues on its activity, we substituted eight amino acid residues with arginine or lysine respectively. All mutants were cloned and expressed in BL21 (DE3) plysS and the mutant proteins were then purified. These mutant versions had higher positive charges but similar structural configurations compared to the wild-type pBD2. Moreover, these mutant proteins showed different antimicrobial activities against E. coli and S. aureus. The mutant I4R of pBD2 had the highest antimicrobial activity. In addition, all the mutants showed low hemolytic activities. Our results indicated that the positively charged residues were not the only factor that influenced antimicrobial activity, but other factors such as distribution of these residues on the surface of defensins might also contribute to their antimicrobial potency.

Molecular cloning, expression and characterization of the porcine ß defensin 2 in E. coli.

Porcine ß defensin 2(pBD2)is a cationic 37-amino acid antimicrobial peptide with disulfide bonds. Synthetic pBD2 had broad antimicrobial activity against pathogenic bacteria, and thus pBD2 could be a good candidate as a bactericidal agent for pigs. This study reported the successful recombinant expression of pBD2 in Escherichia coli and analysis of its antimicrobial activity, its hemolytic activity, salt-tolerance and thermal stability as well. The pBD2 gene, obtained by RT-PCR using the tongue total RNA as a template and cloned into pET30a expression vector, was transformed into E. coli BL21 (DE3) plysS. The recombinant pBD2 was expressed after induction by IPTG and purified by His tag affinity column with 95% purity. The recombinant pBD2 exhibited antimicrobial activity against both Gram-positive S. aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentration (MIC) of recombinant pBD2 against tested bacteria was 10 µg/mL, and the recombinant pBD2 could kill 50% E. coli at 14.39 µg/mL and S. aureus at 21.1 µg/mL. In addition, pBD2 showed low hemolytic activity, salt-tolerance and thermal stability, the properties would be important for its application in practice.

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

Porcine ß defensin 1 (pBD1) is a cationic antimicrobial peptide with three pairs of disulfide bonds. When expressed in insect cells, two polypeptides of different length (pBD1(38) and pBD1(42)) accumulated, which differed by N-terminal truncation. However, only pBD1(42) was found in pigs. pBD1(42) had stronger antimicrobial activity than pBD1(38), and thus could be a good candidate as a bactericidal agent for pigs. In this study, pBD1(42) 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 pBD1(42) was expressed after induction by IPTG and purified by His tag affinity column with 90% purity. The recombinant pBD1(42) 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 pBD1(42) against tested bacteria were 100 µg/mL for E. coli and 80 µg/mL for S. aureus. In addition, pBD1(42) showed low hemolytic activity and high thermal stability. These properties are relevant for the biotechnological applications of the peptide.