PURIFICATION OF HISTIDINE-TAGGED RAS AND ITS USE IN THE DETECTION OF RAS BINDING-PROTEINS

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homoge neity from extracts of E. coli M15 using a one-step procedure which in volved immobilised metal ion chromatography on Ni2+-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/l itre E. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver stai ning of proteins were employed to search for ras-binding proteins pres ent 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 befor e a second round of chromatography on Ni-NTA removed most of these pro teins. Chromatography of a mixture of pre-treated rat brain cytosol an d purified his-ras protein revealed four new protein bands with molecu lar weights of 250, 90, 80 and 70 kDa. These were considered to be can didate ras-binding proteins. It is concluded that the use of his-ras a nd immobilised metal ion chromatography does provide an approach which can be used to identify ras binding proteins present in cellular extr acts.