来自大肠杆菌的M.RsrI DNA甲基转移酶的纯化与特性分析

Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli.

作者信息

Kaszubska W, Webb H K, Gumport R I

机构信息

Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801-3792.

出版信息

Gene. 1992 Sep 1;118(1):5-11. doi: 10.1016/0378-1119(92)90242-h.

DOI:10.1016/0378-1119(92)90242-h

PMID:1511884

Abstract

The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli. Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI. This level of expression represents an approximately 50-fold increase over that present in the natural host. Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity. The purification yielded 100 times more enzyme than was obtained from the same quantity of R. sphaeroides cell paste. M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI. Unlike M.EcoRI, the R. sphaeroides enzyme is a dimer at micromolar concentrations.

摘要

编码来自球形红细菌的RsrI DNA甲基转移酶（M.RsrI）的基因（rsrIM）在大肠杆菌中被克隆并表达。在噬菌体T7启动子的控制下，粗提物中总蛋白的2%是M.RsrI。这种表达水平比天然宿主中的表达水平增加了约50倍。使用DNA纤维素和S-腺苷甲硫氨酸类似物杀稻瘟菌素进行色谱分离，有助于将该酶纯化至同质。纯化得到的酶量比从相同量的球形红细菌细胞糊中获得的酶量多100倍。M.RsrI在每次有效的DNA结合事件中沉积一个甲基基团，其功能但序列不同源的类似物M.EcoRI也是如此。与M.EcoRI不同，球形红细菌的这种酶在微摩尔浓度下是二聚体。