嗜冷芽孢杆菌 M1.BspACI 重组 DNA-甲基转移酶的纯化及其性质

Recombinant DNA-methyltransferase M1.BspACI from Bacillus psychrodurans AC: purification and properties.

机构信息

SibEnzyme, Novosibirsk 630117, Russia.

出版信息

Biochemistry (Mosc). 2010 Dec;75(12):1484-90. doi: 10.1134/s0006297910120096.

DOI:10.1134/s0006297910120096

Abstract

A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5'-CCGC-3') comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5'-CCGC-3'. The kinetic parameters of M1.BspACI DNA methylation are as follows: K(m) for phage λ DNA is 0.053 µM and K(m) for S-adenosyl-L-methionine is 5.1 µM. The catalytic constant (k(cat)) is 0.095 min(-1).

摘要

来自嗜冷芽孢杆菌 AC（识别序列为 5'-CCGC-3'）的限制修饰系统由两个 DNA 甲基转移酶组成：M1.BspACI 和 M2.BspACI。bspACIM1 基因被克隆到 pJW2 载体中，并在大肠杆菌细胞中表达。通过不同载体的色谱法获得了高纯度的 M1.BspACI 制剂。M1.BspACI 的最适温度为 30°C，在 pH8.0 时表现出最大活性。M1.BspACI 修饰识别序列 5'-CCGC-3'中的第一个胞嘧啶。M1.BspACI DNA 甲基化的动力学参数如下：噬菌体 λ DNA 的 K(m)为 0.053 µM，S-腺苷-L-甲硫氨酸的 K(m)为 5.1 µM。催化常数（k(cat)）为 0.095 min(-1)。