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Abstract To make highly expressed swine antimicrobial peptide protegrin-1(PG-1) in prokaryotic expression system, the part of codon of PG-1 was replaced by codon preference of Escherichia coli and a new PG-1 series was designed in this study. Two complementary primers were designed and the whole PG-1 fragment was spliced and amplified by overlap extension PCR. The amplified fragment was inserted into pGEX-4T-1 and the fusion expression plasmid pGEX4T-PG-1 was constructed. The plasmid was transferred into E. coli BL21(DE3) plyS and induced by IPTG (1.0 mmol/L). SDS-PAGE and Western-blotting analysis showed that the recombinant had expressed a 28 kD specific protein in E. coli. By Glutathione Sepharase 4B affinity chromatography, the fusion protein GST-PG-1 was obtained and purified. After cleaved by thrombin, 1.38 mg/L antimicrobial peptide PG-1 was obtained. The recombinant PG-1 showed antibacterial activities against E. coli and Staphylococcus aureus by growth inhibition method.
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Received: 10 March 2010
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