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 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.
Chataway
T K
TK
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Barritt
G J
GJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009532:Nickel / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D018631:ras Proteins / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D009532:Nickel; D018631:ras Proteins