title: Transient and stable expression of the neurotensin receptor NTS1: a comparison of the baculovirus-insect cell and the T-REx-293 expression systems.
abstract: Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [(3)H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs.
authors.affiliation: Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States of America.
authors.forename: Su
authors.initials: S
authors.lastname: Xiao
authors.forename: Jim F
authors.initials: JF
authors.lastname: White
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
authors.forename: Reinhard
authors.initials: R
authors.lastname: Grisshammer
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000818:Animals; D016367:Baculoviridae; D007313:Insecta; D009496:Neurotensin / Q000378:metabolism; D011485:Protein Binding; D018028:Receptors, Neurotensin / Q000378:metabolism*
chemical_list: D018028:Receptors, Neurotensin; D009496:Neurotensin