Larson T J, Ye S Z, Weissenborn D L, Hoffmann H J, Schweizer H
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
J Biol Chem. 1987 Nov 25;262(33):15869-74.
The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.
编码大肠杆菌sn - 甘油3 - 磷酸操纵子阻遏物的glpR基因被克隆到噬菌体λ的强pL启动子下游。这使得在热诱导携带cI857基因的隐性λ溶原菌时，阻遏物能够过量产生。从诱导菌株中纯化出的阻遏物纯度提高了40倍，达到了均一性。纯化方案采用了聚乙烯亚胺和硫酸铵分级分离，随后进行磷酸纤维素和DEAE - 葡聚糖凝胶色谱。通过测量放射性标记诱导物（sn - 甘油3 - 磷酸）与阻遏物的结合来监测纯化过程。经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳评估，纯化后的阻遏物迁移为一条单一的条带，显示其亚基分子量为30,000。在非变性条件下，阻遏物的分子量为100,000 - 130,000，这表明阻遏物在天然条件下是四聚体。使用流动透析研究了阻遏物与sn - 甘油3 - 磷酸的相互作用。对数据进行Scatchard分析表明，每个阻遏物四聚体有四个结合位点，解离常数为31 microM。使用带移电泳研究了阻遏物与DNA的相互作用。阻遏物特异性结合含有glpD、glpK和glpT - A基因控制区域的DNA片段。在sn - 甘油3 - 磷酸存在下，阻遏物与DNA的结合减少。
