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Bacterially Expressed Raf‐1 Catalytic Domain is Highly Associated with GroEL
Author(s) -
Ho Mengfei,
Wilson Brenda A.,
Peterson John W.
Publication year - 1999
Publication title -
journal of the chinese chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.329
H-Index - 45
eISSN - 2192-6549
pISSN - 0009-4536
DOI - 10.1002/jccs.199900101
Subject(s) - groel , chemistry , groes , microbiology and biotechnology , biochemistry , sepharose , protein kinase a , heat shock protein , kinase , enzyme , escherichia coli , biology , gene
Abstract 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‐1, denoted as His 6 ‐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 ELISA analysis revealed a preferential GroEL binding site in the β1 sheet region of the Raf‐1 kinase domain.