Purification of histidine-tagged ras and its use in the detection of ras binding proteins

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    Abstract

    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 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/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.

    Original languageEnglish
    Pages (from-to)167-173
    Number of pages7
    JournalMOLECULAR AND CELLULAR BIOCHEMISTRY
    Volume144
    Issue number2
    DOIs
    Publication statusPublished - 1 Mar 1995

    Keywords

    • histidine-tagged ras
    • immobilized metal ion chromatography
    • metal ion binding proteins
    • Ni-nitriloacetic acid agarose
    • ras
    • ras-binding proteins

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