c-Raf-1 is the initial protein kinase in the growth factor receptor activated MAPK cascade. It has been demonstrated that Raf is associated with Ras, Hsp90, 14-3-3, Mek, Erk, and Src. In order to obtain a constitutively active protein for use in inhibitor design and to avoid contamination with other eukaryotic kinases, the cDNA for the C-terminal catalytic domain (aa 306-648) was subcloned from the mammalian expression vector (pcDNAS-RafBXB) into a bacterial expression vector (pTrcHisB) with an N-terminal Hisfi-affinity handie (PTHB-Raf6244). Following Ni2+-NTA, Sephacryl-300 gel nitration, and CM Sephadex C-50 cation exchange chrornatography, partially purified Raf6244 protein was obtained. Analysis of the Sephacryl-300 column fractions revealed that the bulk of Raf6244 was found in high molecular weight fractions (Mr >300 kDa), did not bind to the CM column, had no kinase activity, and was found to be tighly associated with a major contaminating protein (Mr 57 kDa), which was also present in a similar bacterially expressed GST Raf6244 fusion protein preparation. Native PAGE and western analysis of the high molecular weight fractions indicated that Raf6244 was associated with this 57 kDa protein in a complex with Mr >800 kDa. Microsequencing showed that the first 28 N-tprminal residues of this protein were identical to those of E. coli heat shock protein GroEL. The lower molecular weight fractions containing RafG244 from the Sephacryl-300 column had significantly less GroEL, were able to bind to the CM column, and exhibited kinase activity with c-Raf kinase peptide substrate and Mi'k.
|Original language||English (US)|
|State||Published - 1998|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology