Li Bao-Cun, Zhang Shuang-Quan, Dan Wen-Bing, Chen Yu-Qing, Cao Peng
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, PR China.
Biotechnol Lett. 2007 Jul;29(7):1031-6. doi: 10.1007/s10529-007-9351-4. Epub 2007 Mar 21.
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.
抗菌肽CM4（ABP-CM4）是从中国家蚕中分离得到的一种由35个氨基酸残基组成的阳离子型两亲性α-螺旋肽，具有广泛的抗菌活性。为探索在大肠杆菌中表达ABP-CM4的新方法，通过递归PCR（rPCR）获得的ABP-CM4基因被克隆到载体pET32a中，构建融合表达质粒。融合蛋白Trx-CM4以可溶形式表达，通过镍离子螯合层析纯化，并用甲酸裂解以释放重组CM4。重组CM4通过亲和层析和反相高效液相色谱进行纯化。纯化后的重组肽对大肠杆菌K(12)D(31)、产黄青霉、黑曲霉和小麦赤霉具有抗菌活性。根据抗菌肽数据库（http://aps.unmc.edu/AP/main.html），116种肽含有甲硫氨酸残基，但只有5种肽含有天冬氨酸-脯氨酸位点，这表明在裂解融合蛋白方面，甲酸比溴化氰具有更广泛的应用。ABP-CM4表达的成功应用表明该系统是一种低成本、高效生产毫克级具有生物活性的ABP-CM4的方法。
