One-step purification of bacterially expressed recombinant transducin alpha-subunit and isotopically labeled PDE6 gamma-subunit for NMR analysis.
Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) 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 Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(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 rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.
Guo
Lian-Wang
LW
Department of Pharmacology, University of Wisconsin, Madison, WI 53706, USA. lianwangguo@wisc.edu
Assadi-Porter
Fariba M
FM
Grant
Jennifer E
JE
Wu
Hai
H
Markley
John L
JL
Ruoho
Arnold E
AE
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002846:Chromatography, Affinity; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D005990:Glycerol / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D015293:Transducin / Q000302:isolation & purification* / Q000378:metabolism
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C063842:dnaK protein, E coli; D015293:Transducin; D005990:Glycerol