title: Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles.
abstract: Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay.
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: tho@dartneuroscience.com.
authors.forename: Thao T
authors.initials: TT
authors.lastname: Ho
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: jnguyen@dartneuroscience.com.
authors.forename: Jasmine T
authors.initials: JT
authors.lastname: Nguyen
authors.affiliation: Department of Preclinical Development, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: jupingliu@dartneuroscience.com.
authors.forename: Juping
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: pstanczak@dartneuroscience.com.
authors.forename: Pawel
authors.initials: P
authors.lastname: Stanczak
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: Athompson@dartneuroscience.com.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Thompson
authors.affiliation: Department of Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: gyan@dartneuroscience.com.
authors.forename: Yingzhuo G
authors.initials: YG
authors.lastname: Yan
authors.affiliation: Department of Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: jasmine@dartneuroscience.com.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: callerston@dartneuroscience.com.
authors.forename: Charles K
authors.initials: CK
authors.lastname: Allerston
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: cdillard@dartneuroscience.com.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Dillard
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: hxu@dartneuroscience.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: nschoger@dartneuroscience.com.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Shoger
authors.affiliation: Department of Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: jcameron@dartneuroscience.com.
authors.forename: Jill S
authors.initials: JS
authors.lastname: Cameron
authors.affiliation: Department of Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: emassari@dartneuroscience.com.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Massari
authors.affiliation: Department of Structural Biology, Dart Neuroscience, 12278 Scripps Summit Drive, San Diego, CA 92131, USA. Electronic address: kaertgeerts@dartneuroscience.com.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Aertgeerts
mesh_terms: D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D055550:Protein Stability; D044093:Receptor, Galanin, Type 3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014771:Virion / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D044093:Receptor, Galanin, Type 3; D011993:Recombinant Fusion Proteins
keywords: GPCR protein stabilization; GPCR recombinant protein expression; Membrane protein; Protein engineering; Virus-like particles