Fig. 2 | Journal of Biomolecular NMR

Fig. 2

From: Efficient production of a functional G protein-coupled receptor in E. coli for structural studies

Fig. 2

Fusion protein construct for β1AR expression in E. coli and tests for expression and solubilization optimization. a Fusion protein construct for E. coli expression based on the pRG/III-hs-MBP plasmid (Tucker and Grisshammer 1996). The different sequence parts are labeled for maltose-binding protein (MBP), glycine-serine linker (GS)5, HRV 3C protease cleavage sites (3C, residues LEVLFQ↓GP), NdeI and AgeI restriction sites, thioredoxin A (TrxA), and a deca-histidine tag (H10). be Expression and solubilization tests quantified by western blot. The number under each band represents its relative intensity compared to the strongest band in the gel. b Expression test using different E. coli strains. c Expression test in the absence (−) or in the presence (+) of 20 mg/L cholesterol. d Test of the influence of the expression duration after induction. e Test of membrane solubilization using different detergent mixtures. The solubilized fractions were subjected to nickel-affinity chromatography and eluted by the same buffer for all the samples (20 mM Tris pH 7.5, 350 mM NaCl, 250 mM imidazole, 0.15% DM) before quantification by western blot

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