KpnI DNA甲基转移酶的纯化及该酶与S-腺苷-L-甲硫氨酸的光标记

Purification of the KpnI DNA methyltransferase and photolabeling of the enzyme with S-adenosyl-L-methionine.

作者信息

Finta C, Sulima U, Venetianer P, Kiss A

机构信息

Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.

出版信息

Gene. 1995 Oct 16;164(1):65-9. doi: 10.1016/0378-1119(95)00439-d.

DOI:10.1016/0378-1119(95)00439-d

PMID:7590323

Abstract

An Escherichia coli strain overproducing the KpnI DNA methyltransferase (M.KpnI) was constructed by cloning the kpnIM gene downstream from the inducible T7 phage luminal diameter 10 promoter. A method involving three chromatographic steps has been developed to purify M.KpnI to homogeneity. The purified enzyme has a pH optimum around 7.3 and is inhibited by salts. M.KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet). Photolabeling results from a specific interaction between M.KpnI and AdoMet, as indicated by the dependence of photolabeling on native enzyme conformation and by the inhibitory effect of the AdoMet analogs, sinefungin and S-adenosyl-L-homocysteine (AdoHcy).

摘要

通过将kpnIM基因克隆到可诱导的T7噬菌体腔内直径10启动子下游，构建了一种过量产生KpnI DNA甲基转移酶（M.KpnI）的大肠杆菌菌株。已开发出一种涉及三个色谱步骤的方法，将M.KpnI纯化至同质。纯化后的酶在pH约7.3时具有最佳活性，并受到盐的抑制。在存在S-腺苷-L-[甲基-³H]甲硫氨酸（[甲基-³H]AdoMet）的情况下，M.KpnI可通过紫外线照射进行光标记。光标记是由M.KpnI与AdoMet之间的特异性相互作用引起的，这可通过光标记对天然酶构象的依赖性以及AdoMet类似物、杀稻瘟菌素和S-腺苷-L-高半胱氨酸（AdoHcy）的抑制作用来表明。