Bacterial Expression and One-Step Purification of an Isotope-Labeled Heterotrimeric G-Protein alpha-Subunit
N G. Abdulaev, Xi-Cheng Zhang, Andy Dinh, T Ngo, Phillip Bryan, D M. Brabazon, John Marino, Kevin Ridge
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 heterotrmeric G-protein-subuniG alpha )chimera ( 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.
, Zhang, X.
, Dinh, A.
, Ngo, T.
, Bryan, P.
, Brabazon, D.
, Marino, J.
and Ridge, K.
Bacterial Expression and One-Step Purification of an Isotope-Labeled Heterotrimeric G-Protein alpha-Subunit, Journal of Biomolecular Nmr
(Accessed March 1, 2024)