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