Nwosu V U, Connolly B A, Halford S E, Garnett J
Department of Biochemistry, University of Southampton, UK.
Nucleic Acids Res. 1988 May 11;16(9):3705-20. doi: 10.1093/nar/16.9.3705.
The gene for the Eco RV methylase has been cloned into a plasmid under control of the strong lambda PL promoter and overexpressed in E. coli. This plasmid, pVIC1, gives reliable overexpression of the methylase at levels of about 20% of total protein. Maximum yields of soluble protein are achieved after about 6 hours of induction. If the cells are harvested later than this much of the enzyme is found in the pellet fraction following centrifugation. A two column purification scheme using phosphocellulose and Blue-Sepharose chromatography has been developed. This yielded pure methylase in amounts of 5mg per gram E. coli cell paste. The enzyme is monomeric and methylates the first deoxyadenosine residue in its recognition sequence GATATC.
Eco RV甲基化酶基因已被克隆到一个受强λPL启动子控制的质粒中，并在大肠杆菌中过表达。该质粒pVIC1能可靠地使甲基化酶过表达，其水平约占总蛋白的20%。诱导约6小时后可获得可溶性蛋白的最大产量。如果收获细胞的时间晚于此，离心后会发现沉淀部分中有大量的酶。已开发出一种使用磷酸纤维素和蓝色琼脂糖凝胶色谱的双柱纯化方案。每克大肠杆菌细胞糊可产生5毫克纯甲基化酶。该酶是单体，能使其识别序列GATATC中的第一个脱氧腺苷残基甲基化。
