title: Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.
abstract: Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Marco
authors.initials: M
authors.lastname: Schütz
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Jendrik
authors.initials: J
authors.lastname: Schöppe
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Erik
authors.initials: E
authors.lastname: Sedlák
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hillenbrand
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Nagy-Davidescu
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Janosch
authors.initials: J
authors.lastname: Ehrenmann
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Klenk
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Egloff
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Lutz
authors.initials: L
authors.lastname: Kummer
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019020:Directed Molecular Evolution; D006801:Humans; D008856:Microscopy, Fluorescence; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000235:genetics / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1; D018028:Receptors, Neurotensin; D017473:Receptors, Opioid, kappa; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C114036:neurotensin type 1 receptor