Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.
Li
Bao-Cun
BC
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, PR China.
Zhang
Shuang-Quan
SQ
Dan
Wen-Bing
WB
Chen
Yu-Qing
YQ
Cao
Peng
P
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D012831:Bombyx / Q000737:chemistry* / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D008826:Microbial Sensitivity Tests; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility / Q000187:drug effects
D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins