Bacterially expressed raf-1 catalytic domain is highly associated with GroEL

Raf-1 is a key protein kinase in the mitogen-activated protein kinase cascade. We have subcloned the catalytic domain of Raf-1 into the bacterial expression vectors, pTrcHisB and pGEX-6P-l, denoted as His₆-ΔNRaf and GST-RafBXB, respectively. Chromatography of the recombinant proteins using Ni-NTA agarose, Sephacryl S-300, and glutathione-sepharose revealed association of Raf-1 catalytic domain in a high molecular weight complex with a 57 kDa protein. Microsequencing of this 57 kDa protein identified it as GroEL, a heat shock protein in E. coli important for protein folding. GroEL association with the Raf-1 catalytic domain is specific, as evidenced by its association with both Raf-1 constructs. Native-PAGE gels and Western analysis of gel filtration fractions revealed association of the catalytic domain with a large molecular weight complex consistent with the tetradecameric complex of GroEL. A peptide library of 384 do-decapeptides corresponding to the entire catalytic domain of Raf-1 was constructed by the spot synthesis method. Binding of GroEL and ELIS A analysis revealed a preferential GroEL binding site in the βl sheet region of the Raf-1 kinase domain.