Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli
Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli
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DOI：
10.1074/jbc.272.32.19819
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发表时间：
1997-08-08
影响因子：
4.8
通讯作者：
Leach, DRF
中科院分区：
文献类型：
--
作者：
Connelly, JC;deLeau, ES;Leach, DRF
sbcC和sbcD基因在大肠杆菌中介导回文失活。sbcCD操纵子已被克隆到质粒pTrc 99A中，在强trc启动子的控制下，并被引入携带sbcCD染色体缺失的菌株中。SbcC和SbcD多肽过表达至总细胞蛋白的6%，并且这两种多肽在四步纯化程序中共纯化。纯化的SbcCD是一种进行性双链核酸外切酶，其对Mn 2+具有绝对的需求，并使用ATP作为优选的能量来源。凝胶过滤色谱和沉降平衡分析用于显示SbcC和SbcD多肽在纯化后的某个阶段解离，并且这种解离被Mn 2+的加入逆转。我们证明，SbcD有可能形成一个二级结构基序中发现的一些蛋白磷酸酶，并建议它是一个金属蛋白，包含的SbcCD外切核酸酶的催化中心。
The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli, The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source, Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.