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Purification of histidine-taggedras and its use in the detection ofras binding proteins

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Abstract

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. 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/litreE. 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 forras-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 candidateras-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 identifyras binding proteins present in cellular extracts.

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Abbreviations

his-ras :

histidine-tagged vHa-ras

Ni-NTA:

Ni2+ nitriloacetic acid agarose

IPTG:

isopropyl thio-β-D-galactoside

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Authors and Affiliations

  1. Department of Medical Biochemistry, School of Medicine, Flinders University, G.P.O. Box 2100, 5001, Adelaide, South Australia, Australia

    Timothy K. Chataway & Greg J. Barritt

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  1. Timothy K. Chataway
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  2. Greg J. Barritt
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Chataway, T.K., Barritt, G.J. Purification of histidine-taggedras and its use in the detection ofras binding proteins. Mol Cell Biochem 144, 167–173 (1995). https://doi.org/10.1007/BF00944396

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  • DOI: https://doi.org/10.1007/BF00944396

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