Chataway T K, Barritt G J
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Mol Cell Biochem. 1995 Mar 23;144(2):167-73. doi: 10.1007/BF00944396.
Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts of E. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni(2+)-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litre E. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search for ras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidate ras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identify ras binding proteins present in cellular extracts.
重组组氨酸标签化的v-Ha-ras（his-ras）通过一步法从大肠杆菌M15提取物中纯化至同质，该方法包括在Ni(2+)-次氮基三乙酸琼脂糖（Ni-NTA）上进行固定化金属离子色谱。咪唑洗脱的最佳pH值为6.6，his-ras蛋白的产量（纯度大于95%）约为4mg/升大肠杆菌培养物。将纯化的his-ras与大鼠脑细胞质溶胶的混合物在Ni-NTA上进行色谱分析，并结合蛋白质的SDS-PAGE和银染，以寻找大鼠脑细胞质溶胶中存在的ras结合蛋白。单独将大鼠脑细胞质溶胶在Ni-NTA上进行色谱分析，发现有几种蛋白质不易被咪唑洗脱。这些可能是低丰度的脑金属离子结合蛋白。在第二轮Ni-NTA色谱分析之前，用Ni-NTA对大鼠脑细胞质溶胶进行预处理，去除了大部分这些蛋白质。预处理的大鼠脑细胞质溶胶与纯化的his-ras蛋白混合物的色谱分析显示出四条新的蛋白带，分子量分别为250、90、80和70kDa。这些被认为是候选的ras结合蛋白。结论是，使用his-ras和固定化金属离子色谱确实提供了一种可用于鉴定细胞提取物中存在的ras结合蛋白的方法。
