Kumar S, Cheng X, Pflugrath J W, Roberts R J
Cold Spring Harbor Laboratory, New York 11724.
Biochemistry. 1992 Sep 15;31(36):8648-53. doi: 10.1021/bi00151a035.
The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.
II型DNA（胞嘧啶-5）-甲基转移酶M.HhaI在大肠杆菌中过表达并纯化至表观均一。纯化方案利用独特的高盐反向萃取步骤选择性地溶解M.HhaI，随后进行快速蛋白质液相色谱（FPLC）层析。每克菌泥的纯化蛋白产量为0.75 - 1.0毫克。M.HhaI可以以两种形式分离：与辅因子S-腺苷甲硫氨酸（AdoMet）结合或不含辅因子。结合AdoMet的形式能够在无外源AdoMet的情况下在体外使DNA甲基化。通过对纯化酶的动力学研究，确定了KmAdoMet（60 nM）、KiAdoHye（0.4 nM）和Kcat（0.22 s-1）的值。通过悬滴气相扩散技术使结合辅因子的纯化酶结晶。晶体属于单斜空间群P2(1)，晶胞参数为a = 55.3 Å，b = 72.7 Å，c = 91.0 Å，β = 102.5°，每个不对称单元中有两个M.HhaI分子。这些晶体的衍射极限为2.5 Å以上，适合进行结构测定。
