PURIFICATION AND CHARACTERIZATION OF TRP APO-REPRESSOR
PURIFICATION AND CHARACTERIZATION OF TRP APO-REPRESSOR
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DOI：
10.1073/pnas.80.3.668
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
SIGLER, PB
中科院分区：
文献类型：
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作者：
JOACHIMIAK, A;KELLEY, RL;SIGLER, PB
从携带一个质粒的大肠杆菌高产菌株中分离出均一的色氨酸阻遏蛋白原。该质粒含有色氨酸操纵子串联启动子前的色氨酸R基因（trpR）。采用染料亲和层析和离子交换层析，并结合凝胶电泳分析。当阻遏蛋白与色氨酸操纵子结合时，它可保护一个RsaI限制酶位点不被核酸内切酶切割。从多种浓盐溶液中培养出适合X射线衍射研究的晶体。对未修饰的以及共价交联的阻遏蛋白原进行流体动力学特性和电泳分析表明，游离的阻遏蛋白原等电点为5.9，是一个二聚体，无论是否存在L - 色氨酸，都含有两个相同的12.5千道尔顿亚基。阻遏蛋白 - 操纵子复合物与硝酸纤维素滤膜结合较弱，但限制酶位点保护研究表明，在色氨酸存在的情况下，一个二聚体以表观解离常数<2×10⁻⁹ M结合到操纵子位点。初步的平衡透析实验表明，色氨酸以1.6×10⁻⁵ M的解离常数结合到阻遏蛋白原上。
Homogeneous trp aporepressor was isolated from an overproducing strain of E. coli carrying a plasmid containing trpR preceded by tandem trp operon promoters. Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an RsaI restriction site from endonuclease cleavage. Crystals suitable for X-ray diffraction studies were grown from a variety of concentrated salt solutions. Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing 2 identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan. The repressor operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, 1 dimer is bound to the operator site with an apparent dissociation constant < 2 .times. 10-9 M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 .times. 10-5 M.