Fusion expression of human beta-defensin 3 in E.coli and primary analysis of its antimicrobial activities

Objective: To express human beta-defensin 3 with antimicrobial activities in E.coli. Methods: DNA fragment encoding the mature peptide of human beta-defensin 3 were prepared by PCR with pGEM-T-hBD3 as template and then cloned into pGEX-4T-1. Clones carrying pGEX-4T-1-hBD3 was induced by IPTG. The bacteria were broken down by ultrasonic and the inclusion bodies obtained underwent denaturation, renaturation and purification to obtain GST-hBD3 fusion protein. The fusion protein was cleaved by thrombin to achieve recombinant form hBD3. The antimicrobial activity of the rhBD3 was assayed by minimal inhibitory concentration (MIC) method. Results: rhBD3 was expressed as GST-fusion protein in pGEX-4T-1 prokaryotic expression system. The fusion protein was present in the lysate as inclusion bodies. rhBD3 was released by thrombin digestion and demonstrated a MIC of 4 μg/ml against gram-positive S.aureus and 8 μg/ml against gram-negative E.coli. Conclusion: Recombinant hBD3 with antimicrobial activities was generated in pGEX-4T-1 fusion expression system.