Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line.
In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of approximately 30 microg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device.
Cook
Brian L
BL
Department of Biological Engineering and Center for Biomedical Engineering NE47-379, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Ernberg
Karin E
KE
Chung
Hyeyoun
H
Zhang
Shuguang
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D007668:Kidney; D008322:Mammals; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018035:Receptors, Odorant / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D012243:Rhodopsin / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology
D018035:Receptors, Odorant; C533561:olfactory receptor 17-4, human; D012243:Rhodopsin; D013752:Tetracycline