Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase.
Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase.
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重组杆状病毒表达的小鼠 DNA 甲基转移酶的纯化和表征。
DOI：
10.1006/bbrc.1996.5943
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Reich,NO
中科院分区：
文献类型：
--
作者：
Glickman,JF;Flynn,J;Reich,NO
DNA甲基化对于小鼠的正常胚胎发育至关重要。对 DNA 甲基化如何控制的理解很大程度上取决于 DNA 甲基化系统细胞成分的分离和表征。在真核细胞中甲基化 DNA 的酶是 C-5 胞嘧啶 DNA 甲基转移酶。从历史上看，这种酶的表征一直受到其可用性和纯度的限制。在这里，我们提出了 4 mg 杆状病毒表达的含有镍亲和前导肽的小鼠 DNA 甲基转移酶的一步纯化方法。重组 DNA 甲基转移酶与抑制性 RNA 共纯化，通过核糖核酸酶 A 处理去除抑制性 RNA。与其非重组对应物一样，重组酶通过半甲基化被激活。提出了重组杆状病毒表达的酶与其 MEL 细胞衍生的对应物之间的直接稳态动力学比较。
DNA methylation is essential for normal embryonic development in mice. An understanding of how DNA methylation is controlled is largely dependent upon the isolation and characterization of the cellular components of the DNA methylation system. The enzyme which methylates DNA in eukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availability and purity. Here, we present a single-step purification of 4 mg of baculovirus-expressed mouse DNA methyltransferase containing a nickel-affinity leader peptide. The recombinant DNA methyltransferase co-purified with inhibitory RNA which was removed by treatment with ribonuclease A. Like its non-recombinant counterpart, the recombinant enzyme is activated by hemi-methylation. A direct steady-state kinetic comparison between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented.