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Free keywords:
G protein-coupled receptor; GPCR; Membrane protein; Protein production; Pichia pastoris; Expression system; Optimization; High throughput; Structure determination; X-ray
Abstract:
Recent success in obtaining high-resolution structural data for the first several G proteincoupled receptors (GPCRs) has highlighted the feasibility of structural membrane proteomics approaches for obtaining molecular models of additional GPCRs from among the nearly 800 encoded by the human genome. Yet, production of functional receptors, in general, has proven to be difficult, typically requiring considerable time and cost investments. Here we describe screening, optimization, and scale-up methods we successfully used to produce milligram amounts of functional GPCRs in Pichia pastoris. When we surveyed a large number of receptors recombinantly produced in Pichia, 85% exhibited specific ligand binding, strongly suggesting that this expression system is excellent for producing functional GPCRs. Of the latter group, 20 were optimized according to our protocol. Of these, we produced 10 as milligram amounts of functional receptors using large-scale shaker culture. Cost and time expenditures were considerably lower using the Pichia system than for other successfully employed cell culture systems.
