Preparation of an antibacterial peptide bovine lactoferricin by genetic engineering

摘要

Lactoferricin (Lfcin) is an amphipathic and cationic peptide with strong antibacterial activities against a broad range of Gram-positive, Gram-negative bacteria. It is usually generated by the pepsin-mediated digestion of lactoferrin. This paper investigated the potential for production of bovine lactoferricin (LfcinB) through genetic engineering. Three primers were designed according to amino acid sequence of bovine lactoferricin and then used to construct an oligonucleotide fragment (BeL) encoding lactoferricin. Genetically engineered bacteria (E. coli BL21-pEC- BeL) was successfully constructed after the BeL was inserted into a pEC vector and transformed into E. coli BL21 (DE3). Expression of the pEC vector in E. coli BL21-pEC-BeL was induced by addition of lactose and resulted in production of a fusion protein containing the target peptide LfcinB inside the E. coli cells. Purification of fusion protein was carried out by lysis of bacterial cells with lysozyme, followed by urea precipitation and ethanol precipitation method. The recombinant LfcinB was isolated by acid hydrolysis of the fusion protein to remove the fusion partner and purified through CM52 ion exchange column and Sephadex G-25 column. The molecular weight of the recombinant LfcinB was 3.4KD and its amino acid profile was consistent with the target peptide.