Refolding of G protein α 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 α 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 Gα family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only ∼10% of total Gα protein expressed is active; the rest accumulates in inclusion bodies. Although Giα has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold Gα from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of Gα subunits (human Giα(1), human Gsα(short), human G11α and human Gtα(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 Giα expressed from E. coli showed that although the refolded Gα subunits were soluble and retained partial α-helicity characteristic of the native, folded Gα subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded Giα protein has a native-like secondary structure, but is predominately in a molten globular state.