Nov 20, 2014
High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack posttranslational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the 'test' GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.
Figure: TG nematode lines expressing (b)opsin in both muscles and neurons [M,N] achieve greater receptor expression than TG nematode lines expressing (b)opsin in either muscles [M] or neurons [N]. A) A representative immunoblotted gel. Worms were sonicated and centrifuged to remove debris. Then supernatants of 0.33 µl [M] or 0.67 µl [N], [M,N] (b)opsin worms were loaded on the gels as shown. B) Quantification of immunoblotting results from three independent experiments. Error bars indicate means ±SE. **P<0.01; Student's t test; n=3. C): UV spectrum of (b)isorhodopsin purified from [M,N](b)opsin worms by chromatography and gel filtration. The purity and functionality of the protein is evident from the ratio A280nm/A485nm= 1.52 and its electrophoretic profile (inset). The SDS-PAGE gel was silver stained. Right lane shows the molecular weight standards.
Results from: Salom D, Cao P, Yuan Y, Miyagi M, Feng Z, Palczewski K.Anal Biochem. 2014 Nov 20. pii: S0003-2697(14)00529-6. doi: 10.1016/j.ab.2014.11.008. [Epub ahead of print]