Maehara A, Doi Y, Nishiyama T, Takagi Y, Ueda S, Nakano H, Yamane T
Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
FEMS Microbiol Lett. 2001 Jun 12;200(1):9-15. doi: 10.1111/j.1574-6968.2001.tb10685.x.
A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus (phaZCPR) in Paracoccus denitrificans, was investigated. The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence. The molecular mass was determined to be 93 kDa by size-exclusion chromatography, suggesting that the protein formed a tetramer. A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC--phaP. In a cell-free protein synthesis system using E. coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR. These results suggest that PhaR is a DNA-binding protein and may play a role in the regulation of phaP gene expression.
对在反硝化副球菌聚羟基脂肪酸合成基因座（phaZCPR）中鉴定出的一种假定调节蛋白PhaR进行了研究。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，从重组大肠杆菌中纯化的PhaR蛋白为22 kDa，这与根据核苷酸序列计算出的分子量一致。通过尺寸排阻色谱法测定其分子量为93 kDa，表明该蛋白形成了四聚体。凝胶迁移率变动分析表明，PhaR特异性结合phaC-phaP的基因间区域。在使用大肠杆菌S30提取物的无细胞蛋白质合成系统中，添加纯化的PhaR可抑制phaP基因的表达。这些结果表明，PhaR是一种DNA结合蛋白，可能在phaP基因表达的调控中发挥作用。
