High-efficiency expression of yeast-derived G-protein coupled receptors and 19F labeling for dynamical studies

Abstract

We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2–6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2–4, resulting in final functional production of 0.5–3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media.