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首页> 外文期刊>Protein Expression and Purification >Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease
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Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease

机译：大肠杆菌McrA（一种假定的5-甲基胞嘧啶特异性核酸酶）的克隆，纯化和初步表征

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摘要

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21 (DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn2+ finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dinner with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues. (C) 2008 Elsevier Inc. All rights reserved.
机译：通过将mcrA编码序列克隆到T7启动子后面，可以生产出过量生产大肠杆菌m（5）C McrA限制蛋白的大肠杆菌BL21（DE3）表达菌株。重组mcrA负BL21（DE3）宿主可产生活性McrA，其获得的选择性抑制HpaII甲基化酶体外甲基化DNA的T7噬菌体生长的能力证明了这一点。 mcrA编码区包含几个非最佳大肠杆菌三联体。将pACYC-RIL tRNA编码质粒添加到BL21（DE3）宿主中后，诱导诱导的重组McrA（rMcrA）产量提高了约5至10倍。在37摄氏度下表达的McrA蛋白是不溶的，但在20摄氏度下自动诱导后可回收为可溶性蛋白的很大一部分。rMcrA蛋白质据推测在其推定中包含Cys（4）-Zn2 +指和催化重要的组氨酸三联体核酸酶结构域可与几种金属螯合树脂结合，而无需添加聚组氨酸亲和标签。此功能用于开发一种有效的方案，用于快速纯化近乎均质的rMcrA。通过圆二色性分析测得，天然蛋白质是具有高α-螺旋含量的晚餐。在所有测试的条件下，纯化的rMcrA对HpaII甲基化（Cm（5）CGG）DNA均没有可测量的核酸酶活性，尽管纯化的蛋白确实结合了HpaII甲基化的DNA。这些结果对理解McrA在“限制”含m（5）C的DNA中的体内活性具有启示意义，并暗示rMcrA可作为亲和纯化含m（5）C残基的DNA片段的试剂。 （C）2008 Elsevier Inc.保留所有权利。

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