Matsumura P, Rydel J J, Linzmeier R, Vacante D
J Bacteriol. 1984 Oct;160(1):36-41. doi: 10.1128/jb.160.1.36-41.1984.
We overexpressed the CheY protein by fusing the cheY gene to the tryptophan promoter from Serratia marcescens. Expression of the trp promoter-cheY fusion and subsequent purification of the protein resulted in the isolation of up to 20 mg of homogeneously pure CheY protein from 100 mg of the cytoplasmic supernatant fraction. Purification of the CheY protein was accomplished by exploiting the affinity of CheY protein to cibacron blue dye and molecular sieve chromatography. Preliminary biochemical characterization of the pure CheY protein revealed specific interactions with S-adenosylmethionine and cibacron blue dye. Additional kinetic analysis showed that CheY protein inhibits EcoRI methyltransferase. The amino acid composition of the CheY protein predicted by the DNA sequence of the cheY gene and the amino acid analysis of the CheY protein were in agreement, confirming the authenticity of the purified CheY protein.
我们通过将cheY基因与粘质沙雷氏菌的色氨酸启动子融合来过表达CheY蛋白。色氨酸启动子-cheY融合体的表达以及随后的蛋白质纯化，使得从100 mg细胞质上清液组分中分离出了高达20 mg的纯一的CheY蛋白。CheY蛋白的纯化是利用CheY蛋白与汽巴克隆蓝染料的亲和力以及分子筛色谱法来完成的。对纯CheY蛋白的初步生化特性分析揭示了其与S-腺苷甲硫氨酸和汽巴克隆蓝染料的特异性相互作用。进一步的动力学分析表明，CheY蛋白抑制EcoRI甲基转移酶。由cheY基因的DNA序列预测的CheY蛋白的氨基酸组成与CheY蛋白的氨基酸分析结果一致，证实了纯化的CheY蛋白的真实性。
