Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland.
FEMS Microbiol Lett. 2010 Oct;311(2):126-32. doi: 10.1111/j.1574-6968.2010.02080.x. Epub 2010 Aug 19.
In this study, we describe the characterization, cloning, expression and purification of the lysin A gene of the mycobacteriophage TM4. The gene TM4_gp29 (gp29) is a 1644-bp gene that codes for a 58.6-kDa protein and contains peptidoglycan recognition protein, Zn-binding and amidase catalytic domains. The gene was cloned into Escherichia coli using the 'His-Tag' pQE60 vector. After affinity chromatography-mediated purification, the protein was concentrated and visualized using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Evidence of peptidoglycan-degrading activity was observed initially by a chloroform assay and later by conventional zymogram analysis.
在这项研究中，我们描述了分枝杆菌噬菌体 TM4 的溶菌酶 A 基因的特征、克隆、表达和纯化。基因 TM4_gp29（gp29）是一个 1644 个碱基对的基因，编码一个 58.6kDa 的蛋白质，包含肽聚糖识别蛋白、Zn 结合和酰胺酶催化结构域。该基因使用“His-Tag”pQE60 载体在大肠杆菌中进行克隆。通过亲和层析介导的纯化后，使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳浓缩和观察蛋白质。最初通过氯仿测定法观察到肽聚糖降解活性的证据，随后通过常规酶谱分析进行观察。
