The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line.
High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.
Chelikani
Prashen
P
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Reeves
Philip J
PJ
Rajbhandary
Uttam L
UL
Khorana
H Gobind
HG
D058672:Adrenergic beta-2 Receptor Antagonists; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line / Q000187:drug effects; D002522:Chlorocebus aethiops; D003062:Codon; D006224:Cricetinae; D004082:Dihydroalprenolol / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D008322:Mammals; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013752:Tetracycline / Q000494:pharmacology*
D058672:Adrenergic beta-2 Receptor Antagonists; D000319:Adrenergic beta-Antagonists; D003062:Codon; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D004082:Dihydroalprenolol; D013752:Tetracycline