Large-scale production and study of a synthetic G protein-coupled receptor: human olfactory receptor 17-4.
Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be approximately 50% alpha-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.
Cook
Brian L
BL
Department of Biological Engineering, and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA. cookb@mit.edu
Steuerwald
Dirk
D
Kaiser
Liselotte
L
Graveland-Bikker
Johanna
J
Vanberghem
Melanie
M
Berke
Allison P
AP
Herlihy
Kara
K
Pick
Horst
H
Vogel
Horst
H
Zhang
Shuguang
S
D001681:Biological Assay; D019149:Bioreactors; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000378:metabolism*; D018035:Receptors, Odorant / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D029589:Seminal Plasma Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013057:Spectrum Analysis; D020349:Surface Plasmon Resonance
D003902:Detergents; C473192:OR1D2 protein, human; D043562:Receptors, G-Protein-Coupled; D018035:Receptors, Odorant; D029589:Seminal Plasma Proteins; D002118:Calcium