Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein alpha-subunit.
Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein alpha-subunit (G(alpha)) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G(alpha) chimera (approximately 40 kDa polypeptide) has been tested. The results show that a prodomain fused G(alpha) chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G(alpha) isolated from natural sources. To assay for the functional integrity of the purified G(alpha) chimera at NMR concentrations and probe for changes in the structure and dynamics of G(alpha) that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of G(alpha) obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G(alpha) activation.
Abdulaev
Najmoutin G
NG
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA.
Zhang
Cheng
C
Dinh
Andy
A
Ngo
Tony
T
Bryan
Philip N
PN
Brabazon
Danielle M
DM
Marino
John P
JP
Ridge
Kevin D
KD
D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007553:Isotope Labeling; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry
D009587:Nitrogen Isotopes; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D013381:Subtilisins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D008274:Magnesium