Expression and functional study of an Indolicidin analog in E. coli

• Main Author: 譚德昌
• Other Authors: 毛嘉洪
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/31148216433538584756

碩士 === 國立中興大學 === 獸醫學系 === 90 === Antimicrobial peptides are important components of the innate defenses of mammalians. Indolicidin, a cationic antimicrobial peptide isolated from bovine neutrophils, is a member of cathelicidin family. The action of indolicidin is fast and lethal to a broad spectrum of pathogens. A novel cationic peptide, I-13, based on the primary structure of the indolicidin, has improved activity against Gram-negative bacteria, while it maintains the activity of indolicidin against staphylococci and demonstrates a reduced cytotoxicity. Since I-13 shows impressive in vitro activity against microorganisms, it may be a good candidate for clinical use in humans and animals. In this study, the expression, purification and functional study of I-13 were investigated. DNA fragment encoding I-13 gene was synthesized and inserted into pET32a(+) expression vector and transformed into E. coli BL21(DE3). The I-13 fusion proteins were expressed by induction with 1% lactose and purified by Ni2+-chelating affinity chromatography. The purified I-13 fusion proteins were cleaved by enterokinase to remove fusion partner and the antimicrobial activity of the recombinant I-13 was determined. It was found that the MICs of synthetic I-13 against E. coli (ATCC 25922) and S. aureus (ATCC 25923) were both 8 μM. The MIC of recombinant I-13 against S. aureus (ATCC 25923) was 64 μM and recombinant I-13 could inhibit the growth of E. coli (ATCC 25922) at 128 μM. These results demonstrated that antimicrobial peptide I-13 could be produced by fusion protein technology in prokaryotic expression system.