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. 2006 Jun;15(6):1433-40.
doi: 10.1110/ps.062080006.

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

Prashen Chelikani  1 , Philip J Reeves, Uttam L Rajbhandary, H Gobind Khorana
Affiliations

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

Prashen Chelikani et al. Protein Sci. 2006 Jun.

Abstract

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.

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Figures

Figure 1.
Figure 1.
Nucleotide sequence of the codon-optimized hamster β2-AR gene, and the corresponding amino acid sequence. The locations of the restriction sites in the gene are shown above the DNA sequence. The synthetic gene contains at its C terminus the rhodopsin C-terminal octapeptide sequence (underlined) to facilitate detection and purification of the protein using the monoclonal antibody rho-1D4.
Figure 2.
Figure 2.
Immunoblot analysis of β2-AR using the monoclonal antibody rho-1D4 (the epitope tag for this antibody was added to the C-terminal tail of β2-AR gene). β2-AR transiently expressed in HEK293S (lane A). By using a HEK293S (GnT−) cell line defective in N-acetylglucosamine transferase I, β2-AR was expressed with restricted and homogeneous N-glycosylation (lane B). Mobility of molecular weight standards is indicated on the left of the gel.
Figure 3.
Figure 3.
Saturation binding assays for membrane bound β2-AR from COS-1 (A), and HEK293S-TetR stable cell line (B) were performed using [3H] dihydroalprenolol as the radioligand (TRK 649, Amersham). Saturation assays were performed using 0.1–20 nM [3H] DHA, and 2–20-μg membrane protein/well. Binding of [3H] DHA in the presence of 10 μM alprenolol was used as a measure of nonspecific binding. Specific binding was derived by subtracting nonspecific binding from the total binding. Saturation binding data were obtained from a minimum of two independent determinations done in triplicate, and are shown as mean ± SE. Nonspecific binding was always <10% of total binding. Data were analyzed by nonlinear regression using PRISM software (Graph Pad Software).
Figure 4.
Figure 4.
SDS-PAGE (10%) of the solubilized (lane A) and rho-1D4-affinity purified β2-AR (lane B). Proteins were detected by Coomassie staining. (Lane A) 5 μL of solubilized membrane protein, (lane B) 5 μL of rho-1D4-purified protein (25 pmol of receptor), (lane M) molecular weight standards. The receptor produced in HEK293S-TetR was glycosylated and migrated as two bands on the SDS-PAGE with the minor band around 47 kDa and the major band showing an apparent molecular mass of around 60 kDa.
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