title: Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1.
abstract: G protein-coupled receptors (GPCRs) are associated with a wide array of diseases and are targets of most of the medicines sold worldwide. Despite their clinical importance, only 25 unique GPCR structures have been determined as of April 2014. The first step for structural studies is to establish the expression of correctly folded, functional receptors in recombinant host cells at quantities to allow subsequent purification and crystallization trials. Here we describe the T-REx™-inducible expression system to construct and select a stable HEK293 cell line for high-level expression of functional neurotensin receptor type I (NTSR1). We also present the protocols used for the adaptation of the cells into suspension culture, as well as the optimization of the induction parameters for NTSR1 expression, which led to 1 mg of purified NTSR1 per liter suspension culture in bioreactors.
authors.affiliation: Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Su
authors.initials: S
authors.lastname: Xiao
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
authors.forename: Reinhard
authors.initials: R
authors.lastname: Grisshammer
mesh_terms: D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D018703:Founder Effect; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D018028:Receptors, Neurotensin / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; C114036:neurotensin type 1 receptor; D013752:Tetracycline