High-level expression and purification of the human bradykinin B2 receptor in a tetracycline-inducible stable HEK293S cell line

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Abstract

The B2 bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B2 receptor for future biophysical studies. Different tagged B2 receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B2 receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60 pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B2 receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B2 receptor with tetracycline and sodium butyrate led to a level of 100 pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-β-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B2 receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.

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Materials

BK was purchased from Sigma, myo-[2-3H] inositol (16 Ci/mmol) and (prolyl 2,4-3,4-3H) bradykinin ([3H]BK) (40–100 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Hydroxyphenyl-propionyl-HOE-140 (HPP-HOE-140) was kindly supplied by Professor J. Martinez (CNRS, Montpellier, France); it was radioiodinated using [125I] Na (2000 Ci/mmol) and IODO-GEN as oxidizing agent. HEK293S and HEK293S TetR cells expressing the Tet operon repressor protein were kindly provided by P.J. Reeves (present

Ligand-binding properties of tagged B2 receptor constructs

In order to evaluate the influence of the tags on ligand recognition and expression level of the receptor, the different tagged receptor constructs were transiently expressed in HEK293S. As schematically represented in Fig. 1a, tags were inserted at the N- and/or C-terminus of the B2 receptor truncated at the Asn3 glycosylation site. Indeed, suppression of the potential glycosylation site might favor tag recognition and had no significant influence on the pharamocological properties of the

Discussion

In this study, the overexpression and purification of the human bradykinin B2 receptor is described. We decided to overexpress in HEK293S cells for several reasons: (1) mammalian cells offer the advantages of posttranslational modification and proper folding of GPCRs [9], [11], [31], (2) HEK293S cells can be grown adherently, but also can be easily adapted to growth in suspension culture thus allowing large scale production in a bioreactor [14], (3) HEK293S cells have been demonstrated to be a

Acknowledgments

This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Ministère de la Recherche (ACI “Molécules et Cibles Thérapeutiques” n°355) and the Fondation pour la Recherche Médicale. We are grateful to Jean-François Guichou and Alain Chavanieu for the synthesis of the C9 peptide. We thank Dr. Philip J. Reeves (University of Essex, Colchester, UK) for the generous gift of HEK293S TetR cells.

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