Production of bioactive porcine antibacterial peptide PR-39 in Escherichia coli using SUMO fusion technology
Volume : 54 Page : 9-19
ZW Chen, SL Hsuan, JH Lin, WZ Huang, HJ Lin, JP Wang
PR-39 is an antibacterial peptide that could be isolated from porcine small intestine, neutrophils, and spleen. The peptide is a proline-arginine-rich peptide with 39 amino acid residues. Previous studies showed that PR-39 exhibits antibacterial activity against several Gram-positive and Gram-negative bacteria. In addition to its antibacterial properties, PR-39 also exerted other biological activities such as regulation of angiogenesis, promotion of wound repair, and inhibition of cancer cell invasion and metastasis. The objective of this study is to establish a method for production of recombinant RP-39 (rPR-39) using Escherichia coli in order to obtain rPR-39 for further evaluating its potential applications as therapeutic agent and feed additive. The DNA sequence encoding the PR-39 was designed according to E. coli preferred codons and synthesized through overlapping extension polymerase chain reaction. The codon-optimized PR-39 gene was inserted into an expression vector which contains the small ubiquitin-like modifier (SUMO) gene for construction of fusion expression vector, and then this vector was transformed into E. coli. Recombinant His-tagged SUMO-PR-39 (rSUMO-PR-39) fusion protein was expressed in soluble form in E. coli and could be purified by immobilized metal ion affinity chromatography. After the rSUMO-PR39 fusion protein was cleaved by the SUMO protease, released rPR-39 was further purified by gel filtration. The typical yield of rPR-39 was 55 μg with purity above 95% from 1 L culture. The rPR-39 showed antibacterial activity against E. coli with a minimum inhibitory concentration of 0.2 mg/mL. This study has established a method for production of rPR-39. The optimal conditions for production of rPR-39 will be further determined.