Department of Bioengineering and Technology, Kangwon National University, Chuncheon 200-701, Korea.
J Microbiol Biotechnol. 2010 Feb;20(2):350-5.
Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.
在人类抗菌肽 (hAMPs) 中，DCD-1L 在宽 pH 范围和高盐浓度下对广谱抗菌活性。它为传统抗生素提供了一种有前途的替代品。使用人胎儿 cDNA 文库作为模板，通过 PCR 扩增了 458bp 长的 dermcidin cDNA。编码一个额外甲硫氨酸残基的 MDCD-1L 基因的 147bp 片段亚克隆到 pTYB11 载体中。重组 MDCD-1L 在大肠杆菌中表达为内含肽融合蛋白，然后通过使用几丁质珠的亲和层析进行纯化。通过三氯乙酸凝胶电泳检测到约 5kDa 的分子量的小肽。从凝胶中纯化重组 MDCD-1L 肽，并通过纳升液相色谱-电喷雾-串联质谱 (nanoLC-ESI-MS/MS) 分析确定其氨基酸序列。起始氨基酸甲硫氨酸仍连接在重组 MDCD-1L 的 N 端区域。纯化的 MDCD-1L 对微球菌测试菌株表现出抗菌活性。
