Nwosu V U
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Biochem J. 1992 May 1;283 ( Pt 3)(Pt 3):745-50. doi: 10.1042/bj2830745.
The gene coding for Escherichia coli dam methylase was isolated from a dam+ K12 strain by the PCR method. The gene was subcloned into an overexpression vector under the control of the strong lambda PL promoter. The resultant construct produced the dam methylase at about 20% of total cellular protein. Purification of the protein was achieved with two chromatography columns and yielded 6 mg of pure methylase per gram cell paste. The methylase readily methylates the synthetic dodecamer GACTGATCAGTC containing its recognition sequence (underlined). It also methylates a synthetic dodecamer containing the EcoRV recognition sequence GATATC. However, methyl transfer is to the second adenine in the EcoRV sequence.
通过聚合酶链反应（PCR）方法从 dam⁺ K12 菌株中分离出编码大肠杆菌 dam 甲基化酶的基因。该基因被亚克隆到在强λPL 启动子控制下的过表达载体中。所得构建体产生的 dam 甲基化酶约占细胞总蛋白的 20%。通过两根色谱柱实现了该蛋白的纯化，每克细胞糊可产生 6 毫克纯甲基化酶。该甲基化酶很容易使含有其识别序列（下划线部分）的合成十二聚体 GACTGATCAGTC 甲基化。它还能使含有 EcoRV 识别序列 GATATC 的合成十二聚体甲基化。然而，甲基转移是在 EcoRV 序列中的第二个腺嘌呤上。
