Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease.
Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease.
复制标题
DOI：
10.1016/j.pep.2008.06.016
复制
发表时间：
2008-11
影响因子：
1.6
通讯作者：
Dunn JJ
中科院分区：
文献类型：
--
作者：
Mulligan EA;Dunn JJ
用高效表达大肠杆菌BL 21（DE 3）的方法，通过将mcrA编码序列克隆到T7启动子后面来产生大肠杆菌m5 C McrA限制性蛋白。重组mcrA减去BL 21（DE 3）主机产生活性McrA证明了其获得的能力，选择性地限制T7噬菌体的生长含有DNA甲基化在体外由HpaII甲基化酶。mcrA编码区含有几个非最佳E.大肠杆菌三联体。将编码pACYC-RIL tRNA的质粒添加到BL 21（DE 3）宿主中，诱导后重组McrA（rMcrA）的产量增加约5至10倍。在37 °C下表达的McrA蛋白是不溶的，但在20 °C下自诱导后，回收了显著部分作为可溶性蛋白。rMcrA蛋白被预测在其推定的核酸酶结构域中含有Cys 4-Zn 2+指和催化重要的组氨酸三联体，其结合到几种金属螯合树脂而不添加聚组氨酸亲和标签。这一特点被用来开发一个有效的协议，快速纯化几乎均匀的rMcrA。通过圆二色性分析测量，天然蛋白质是具有高α-螺旋含量的二聚体。在所有测试条件下，纯化的rMcrA对HpaII甲基化（Cm 5CGG）DNA没有可测量的核酸酶活性，尽管纯化的蛋白质确实特异性结合HpaII甲基化DNA。这些结果的影响，了解在体内活动的McrA在“限制”m5 C-含有DNA，并建议rMcrA可能有效用作为试剂的亲和纯化的DNA片段含有m5 C残基。
Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m5C 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 °C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 °C. rMcrA protein, which is predicted to contain a Cys4-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 dimer with a high α-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm5CGG) 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” m5C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m5C residues.
登录
查看更多内容
影响因子：
3.2
作者：
WAITEREES, PA;KEATING, CJ;BENNER, JS
通讯作者：
BENNER, JS
影响因子：
3.5
作者：
Baylin, SB;Esteller, M;Herman, JG
通讯作者：
Herman, JG
影响因子：
3.2
作者：
Anton, BP;Raleigh, EA
通讯作者：
Raleigh, EA
影响因子：
5
作者：
Rauch, T;Pfeifer, GP
通讯作者：
Pfeifer, GP
影响因子：
14.9
作者：
RALEIGH, EA;MURRAY, NE;HANAHAN, D
通讯作者：
HANAHAN, D