title: Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity.
abstract: We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
authors.forename: Igor
authors.initials: I
authors.lastname: Dodevski
authors.forename: Manca
authors.initials: M
authors.lastname: Kenig
authors.forename: Stefan
authors.initials: S
authors.lastname: Dudli
authors.forename: Anja
authors.initials: A
authors.lastname: Mohr
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Hermans
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D009154:Mutation; D010843:Pichia; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D018028:Receptors, Neurotensin / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic
chemical_list: D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor