Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.
High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.
Kaiser
Liselotte
L
Center for Biomedical Engineering, NE47-379, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, USA.
Graveland-Bikker
Johanna
J
Steuerwald
Dirk
D
Vanberghem
Mélanie
M
Herlihy
Kara
K
Zhang
Shuguang
S
D002021:Buffers; D002474:Cell-Free System / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D003902:Detergents; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis; D018035:Receptors, Odorant / Q000096:biosynthesis*; D029589:Seminal Plasma Proteins / Q000096:biosynthesis
D002021:Buffers; D003902:Detergents; D008565:Membrane Proteins; C473192:OR1D2 protein, human; D043562:Receptors, G-Protein-Coupled; D018035:Receptors, Odorant; D029589:Seminal Plasma Proteins