A screening system for the identification of refolding conditions for a model protein kinase, p38alpha.
Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38alpha, and applied this system to denatured p38alpha derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38alpha can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38alpha protein kinase.
Cowan
Richard H
RH
Department of Biological Sciences, Gibbet Hill Road, University of Warwick, Coventry CV4 7AL, UK.
Davies
Rick A
RA
Pinheiro
Teresa T J
TT
D002850:Chromatography, Gel; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology; D048308:Mitogen-Activated Protein Kinase 14 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D048308:Mitogen-Activated Protein Kinase 14