Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, Jiangsu, China.
Amino Acids. 2010 Nov;39(5):1545-52. doi: 10.1007/s00726-010-0625-0. Epub 2010 May 29.
Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with Npro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni2+-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.
抗菌肽 CM4 是一种具有广谱抗菌、抗真菌和抗肿瘤活性的小阳离子肽。已经开发了多种使用重组技术生产小抗菌肽的策略。迄今为止，尚未有报道努力获得大量活性重组 CM4。为了建立基于细菌的 CM4 生产系统，将 CM4 克隆到 pET28a 中，并与 Npro 突变体（EDDIE）融合表达。表达的 CM4 作为 EDDIE 沉淀为包涵体。通过从化学变向到反化学变向的体外重折叠，融合伴侣通过自切割从自体蛋白酶的 C 末端释放，使 CM4 蛋白具有真实的 N 末端。纯化的 CM4 通过 Ni2+-螯合层析柱和阳离子交换层析柱进行分离。质谱分析表明该蛋白的分子量为 4132.56 道尔顿，与理论预期的分子量相等。CM4 的 N 端测序表明其序列与天然蛋白相对应。重组 CM4 表现出与先前报道相同的抗菌和抗肿瘤活性。本研究中提出的表达策略允许方便地以高产量和容易地纯化具有天然序列的重组 CM4。
