Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, <Emphasis Type="Italic">Bombyx mori</Emphasis> |
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Authors: | Bao-Cun Li Shuang-Quan Zhang Wen-Bing Dan Yu-Qing Chen Peng Cao |
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Institution: | (1) Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, 210097, PR China;(2) Laboratory of Molecular Medicine, Jiangsu Province Institute of Traditional Chinese Medicine, Jiangsu, Nanjing, 210028, PR China |
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Abstract: | The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-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 Ni2+-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 K12D31, 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. |
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Keywords: | Antibacterial peptide CM4 Esherichia coli Formic acid Fusion expression Purification |
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