Background Human β-defensin-3(HBD_3)is an epithelial peptide that has been demonstrated to have a salt-insensitivebroad spectrum of potent antimicrobial activity.Expressing antimicrobial peptides in Escherichia coli(E.coli)is verydifficult for it can result in death of the bacterial host cells.Our aim was to establish a prokaryotic system expressingsoluble HBD_3 protein and demonstrate the antimicrobial activity of the expressed protein.We then studied whether thehost cells would activate the suicide pathways.Methods We first cloned the complementary DNA coding for the mature chain of HBD_3,inserted it into the vectorPGEX-KG then transformed E.coli BL21(DE3)with the appropriate recombinant plasmid.After induction with 0.5 mmol/Lisopropyl-1-thio-β-D-galactopyranoside(IPTG)the transformed E.coli produced a recombinant glutathione S-transferaseand HBD_3(GST-HBD_3)fusion protein.The fusion protein was treated with thrombin to produce pure HBD_3 protein thenthe antimicrobial activity of HBD_3 was evaluated in a liquid microdilution assay.Results The fusion protein GST-HBD_3 was efficiently cleaved by thrombin and yielded HBD_3 that hadanti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 μg/ml.The E.coli strainexpressing the recombinant protein did not grow slower than the empty vector strain.Conclusion Active HBD_3 in E.coli by expressing the recombinant protein GST-HBD_3 could be produced,and suicidedid not occur in the E.coli strain expressing the recombinant protein. Background Human β-defensin-3 (HBD_3) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E.coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressingsoluble HBD_3 protein and demonstrate the antimicrobial activity of the expressed protein. We then studied either the host cells would activate the suicide pathways. Methods We first cloned the complementary DNA coding for the mature chain of HBD_3, inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol / Lisopropyl-1-thio-β-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD_3 (GST-HBD_3) fusion protein. The fusion protein was treated with thrombin to produce pure HBD_3 protein thenthe antimicrobial activity of H BD_3 was evaluated in a liquid microdilution assay. Results of fusion protein GST-HBD_3 was efficiently cleaved by thrombin and yielded HBD_3 that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 μg / ml. The E. coli strainexpressing the recombinant protein did not grow slower than the empty vector strain. Confocal Active HBD_3 in E. coli by expressing the recombinant protein GST-HBD_3 could be produced, and suicidedid not occur in the E. coli strain expressing the recombinant protein.