荚膜红细菌MopB DNA结合结构域的表达、纯化、结晶及初步X射线分析
Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of Rhodobacter capsulatus MopB.
作者信息
Müller Alexandra, Schlicker Christine, Fehringer Maria, Masepohl Bernd, Hofmann Eckhard
机构信息
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
出版信息
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Mar 1;67(Pt 3):377-9. doi: 10.1107/S1744309110054710. Epub 2011 Feb 25.
The LysR-type regulator MopB represses transcription of several target genes (including the nitrogen-fixation gene anfA) in Rhodobacter capsulatus at high molybdenum concentrations. In this study, the isolated DNA-binding domain of MopB (MopBHTH) was overexpressed in Escherichia coli. Purified MopBHTH bound the anfA promoter as shown by DNA mobility-shift assays, demonstrating the function of the isolated regulator domain. MopBHTH was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M lithium sulfate, 0.1 M phosphate/citrate pH 4.2, 20%(w/v) PEG 1000 at 291 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=61.84, c=139.64 Å, α=β=90, γ=120°, and diffracted to 3.3 Å resolution at a synchrotron source.
LysR 型调控因子 MopB 在高钼浓度下抑制荚膜红细菌中几个靶基因（包括固氮基因 anfA）的转录。在本研究中，MopB 的分离 DNA 结合结构域（MopBHTH）在大肠杆菌中过表达。如 DNA 迁移率变动分析所示，纯化的 MopBHTH 结合 anfA 启动子，证明了分离的调控结构域的功能。采用坐滴气相扩散法，在 291 K 下，于含有 0.2 M 硫酸锂、0.1 M 磷酸盐/柠檬酸盐（pH 4.2）、20%（w/v）聚乙二醇 1000 的条件下，使 MopBHTH 结晶。晶体属于空间群 P3(1)21 或 P3(2)21，晶胞参数 a = b = 61.84，c = 139.64 Å，α = β = 90°，γ = 120°，在同步辐射源处衍射至 3.3 Å 分辨率。
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