Patel J, Taylor I, Dutta C F, Kneale G, Firman K
Biophysics Laboratories, School of Biological Sciences, Portsmouth Polytechnic, U.K.
Gene. 1992 Mar 1;112(1):21-7. doi: 10.1016/0378-1119(92)90298-4.
We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.
我们已将编码I型DNA甲基转移酶（MTase）M.EcoR124的两个亚基（HsdM和HsdS）的基因克隆到专门构建的表达载体pJ119中。这些亚基已作为完整的MTase一起合成。我们还在T7基因10启动子或lacUV5启动子的控制下克隆了单个亚基编码基因。在所有情况下均获得了高水平的表达。虽然发现HsdM是可溶的，但HsdS是不可溶的。然而，在共同产生的HsdM亚基存在的情况下，发现HsdS作为活性MTase的一部分存在于可溶部分中。我们已对克隆的多亚基酶进行了部分纯化，并表明它在体内和体外均能够进行DNA甲基化。
