Nb35 expression and purification

The production and purification of Nb35 were performed following a protocol established by Kobilka and co-workers (18). Nb35 having a C-terminal 6His-tag was expressed in the periplasm of Escherichia coli strain BL21 following induction with 1 mM isopropyl-β-d-thiogalactopyranoside. Cultures of 2L were grown to an optical density at 600 nm of 0.6 at 37°C in LB medium containing 0.1% glucose and ampicillin (100 μg/ml). Induced cultures were grown overnight at 25°C. Cells were harvested by centrifugation and lysed in ice-cold buffer [50 mM tris-HCl (pH 8), 125 mM sucrose, and 2 mM EDTA]. Lysate was centrifuged to remove cell debris, and Nb35 was purified by nickel affinity chromatography. Eluate was concentrated to 5 mg/ml and loaded onto a Superdex 200 (16/600 column, GE Healthcare) at a 1 ml/min of flowrate. Fractions containing the monodisperse peak of Nb35 were pooled and dialyzed overnight against a buffer containing 10 mM Hepes (pH 7.5) and 100 mM NaCl at room temperature (RT). The dialyzed sample was concentrated to approximately 100 mg/ml using a 10-kDa MWCO concentrator (Millipore). Aliquots were stored at −80°C until use.