F 质粒 RepE 的表达与纯化及其与操纵子 DNA 复合物的初步 X 射线晶体学研究。
Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNA.
作者信息
Nakamura Akira, Wada Chieko, Miki Kunio
机构信息
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
出版信息
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Apr 1;63(Pt 4):346-9. doi: 10.1107/S1744309107012894. Epub 2007 Mar 30.
Abstract
The replication initiator factor RepE of the F plasmid in Escherichia coli is an essential protein that stringently regulates the F-plasmid copy number. The RepE protein has a dual function: its monomer functions as a replication initiator, while its dimer acts as a transcriptional repressor of the repE gene. The wild-type dimeric RepE protein was expressed as an N-terminal histidine-tagged protein, purified under native conditions with a high salt concentration and crystallized in complex with the repE operator DNA using the sitting-drop vapour-diffusion technique. The crystals diffracted to a resolution of 3.14 A after the application of dehydration and crystal annealing and belong to space group P2(1), with unit-cell parameters a = 60.73, b = 99.32, c = 95.00 A, beta = 108.55 degrees.
摘要
大肠杆菌中F质粒的复制起始因子RepE是一种必需蛋白，它严格调控F质粒的拷贝数。RepE蛋白具有双重功能：其单体作为复制起始因子，而其二聚体作为repE基因的转录抑制因子。野生型二聚体RepE蛋白表达为N端带组氨酸标签的蛋白，在高盐浓度的天然条件下纯化，并使用坐滴气相扩散技术与repE操纵子DNA形成复合物结晶。在进行脱水和晶体退火处理后，晶体的衍射分辨率达到3.14 Å，属于空间群P2(1)，晶胞参数为a = 60.73、b = 99.32、c = 95.00 Å，β = 108.55°。
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