Gross M, Sweet R W, Sathe G, Yokoyama S, Fasano O, Goldfarb M, Wigler M, Rosenberg M
Mol Cell Biol. 1985 May;5(5):1015-24. doi: 10.1128/mcb.5.5.1015-1024.1985.
The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.
全长正常和T24突变型人H-ras蛋白以及T24突变体的两种截短衍生物在大肠杆菌中高效表达。这些蛋白积累至细胞总蛋白的1%至5%，并且每种都能被抗ras单克隆抗体特异性识别。两种全长蛋白以及一种羧基末端截短衍生物（缺失23个氨基酸残基）在细胞裂解后可溶，并且无需使用变性剂即可纯化至90%的纯度。相比之下，一种氨基末端截短的ras衍生物（缺失22个氨基酸残基）需要用尿素处理才能溶解。通过蛋白质印迹上的配体结合、免疫沉淀和标准滤膜结合程序相结合的方法评估了这四种蛋白的鸟嘌呤核苷酸结合活性。全长蛋白表现出相似的结合动力学，化学计量比接近每摩尔蛋白结合1摩尔GTP。它们表现出相似的结合动力学，化学计量比接近每摩尔蛋白结合1摩尔GTP。羧基末端截短的蛋白也能结合GTP，但程度降低，而氨基末端截短的蛋白没有结合活性。显然，ras的羧基末端结构域虽然对转化功能很重要，但在GTP结合中并不起关键作用。
