One-step purification of bacterially expressed recombinant transducin α-subunit and isotopically labeled PDE6 γ-subunit for NMR analysis

Interactions between the transducin α-subunit (Gαt) and the cGMP phosphodiesterase γ-subunit (PDEγ) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Gαt/PDEγ complexes, in particular, the signaling-state complex represented by Gαt·GTPγS/PDEγ. As a preliminary step in our effort for NMR structural analysis of Gαt/PDEγ interactions, we have developed efficient protocols for the large-scale production of recombinant Gαt (rGαt) and homogeneous and functional isotopically labeled PDEγ from Escherichia coli cells. One-step purification of rGαt was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGαt. The purified rGαt was found to be functional in GTPγS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEγ, rGαt·GTPγS/[U-13C,15N]PDEγ. The labeled PDEγ sample yielded a well-resolved 1H–15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGαt and isotope-labeled PDEγ should support further NMR structural analysis of the rGαt/PDEγ complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.