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

• Published in: Protein expression and purification 2007-02, Vol.51 (2), p.187-197
• Main Authors: Guo, Lian-Wang, Assadi-Porter, Fariba M., Grant, Jennifer E., Wu, Hai, Markley, John L., Ruoho, Arnold E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Carbon Isotopes; Chromatography, Affinity; Cyclic Nucleotide Phosphodiesterases, Type 6; DnaK; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - isolation & purification; Glycerol - pharmacology; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; HSP70 Heat-Shock Proteins - isolation & purification; HSQC; Isotope Labeling; Isotope-labeled protein; Molecular Sequence Data; Nitrogen Isotopes; NMR; Nuclear Magnetic Resonance, Biomolecular; One-step purification; PDE6 γ-subunit; Phosphoric Diester Hydrolases - isolation & purification; Phosphoric Diester Hydrolases - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant transducin α-subunit; Signal Transduction; Transducin - isolation & purification; Transducin - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.07.012

Interactions between the transducin α-subunit (Gα t) 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 Gα t/PDEγ complexes, in particular, the signaling-state complex represented by Gα t·GTPγS/PDEγ. As a preliminary step in our effort for NMR structural analysis of Gα t/PDEγ interactions, we have developed efficient protocols for the large-scale production of recombinant Gα t (rGα t) and homogeneous and functional isotopically labeled PDEγ from Escherichia coli cells. One-step purification of rGα 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 rGα t. The purified rGα t 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, rGα t·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 rGα t and isotope-labeled PDEγ should support further NMR structural analysis of the rGα t/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.