Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

Abstract

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and ^(15)N_2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2/L of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two ^(15)N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of ^(15)N chemical shifts strongly suggests that the ^(15)N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotopelabeled G protein-coupled receptor by bacterial fermentation.

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