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 [3H] 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.
Highlights
G protein-coupled receptors (GPCRs) are integral membrane proteins that play a central role in cell signaling by transmitting extracellular signals across membranes to intracellular effector pathways
NTS1and NTS2 belong to the class A GPCR family, whereas NTS3 is a member of the sortilin family with a single transmembrane domain [11,12,13]
We provide a quantitative comparison between the two production hosts regarding aspects of functional neurotensin receptor 1 (NTS1) expression levels and receptor yield after purification, as well as binding properties and cell surface display of the receptors
Summary
G protein-coupled receptors (GPCRs) are integral membrane proteins that play a central role in cell signaling by transmitting extracellular signals across membranes to intracellular effector pathways. As of December 2012, structures of 16 unique GPCRs are available (http://blanco.biomol.uci.edu/mpstruc/listAll/list), including the structure of the neurotensin receptor 1 bound to its peptide agonist [2]. Neurotensin (NT) is a 13 amino acid residue peptide that is found in the nervous system and in peripheral tissues [5]. NT displays a wide range of biological activities and plays important roles in Parkinson’s disease, in pathogenesis of schizophrenia, in modulation of dopamine neurotransmission, hypothermia, antinociception and in promoting the growth of cancer cells [6,7,8,9,10]. Most of the known effects of NT are mediated through NTS1 [9]
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