Refolding of G protein alpha subunits from inclusion bodies expressed in Escherichia coli.
Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The alpha subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G(alpha) family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only approximately 10% of total G(alpha) protein expressed is active; the rest accumulates in inclusion bodies. Although G(ialpha) has been readily expressed in soluble form (to 10 mg/L), other alpha subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G(alpha) from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G(alpha) subunits (human G(ialpha(1)), human G(salpha(short)), human G(11alpha) and human G(talpha(cone))), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G(ialpha) expressed from E. coli showed that although the refolded G(alpha) subunits were soluble and retained partial alpha-helicity characteristic of the native, folded G(alpha) subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G(ialpha) protein has a native-like secondary structure, but is predominantly in a molten globular state.
McCusker
Emily
E
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Robinson
Anne Skaja
AS
D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet
D044385:GTP-Binding Protein alpha Subunits