Interactions between the transducin alpha-subunit (G alpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDE gamma) 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 G alpha(t)/PDE gamma complexes, in particular, the signaling-state complex represented by G alpha(.)(t)GTP gamma S/PDE gamma. As a preliminary step in our effort for NMR structural analysis of G alpha(t)/PDE gamma interactions, we have developed efficient protocols for the large-scale production of recombinant G alpha(t) (rG alpha(t)) and homogeneous and functional isotopically labeled PDE gamma from Escherichia coli cells. One-step purification of rG alpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rG alpha(t). The purified rG alpha(t) was found to be functional in GTP gamma S/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDE gamma, rG alpha(.)(t)GTP gamma S/[U-C-13, N-15]PDE gamma. The labeled PDE gamma sample yielded a well-resolved H-1-N-15 HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rG alpha(t) and isotope-labeled PDE gamma should support further NMR structural analysis of the rG alpha(t)/PDE gamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins. (c) 2006 Elsevier Inc. All rights reserved.