Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris
Section snippets
Materials
Pichia pastoris strain SMD 1163 was obtained from Invitrogen (San Diego, California, USA), components of Yeast growth media were purchased from Difco (MD, USA) and Sigma (Taufkirchen, Germany). G418 was from Calbiochem (Dermstadt, Germany), DMSO was from Roth (Karlsruhe, Germany) and Glass beads (0.5 mm) were from Euler Prozesstechnik (Frankfurt, Germany). [3H]Bradykinin (60–90.0 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA, USA). Restriction enzymes, PNGaseF and EndoH were
Recombinant construct for expression of the B2R
For the production of human B2R in P. pastoris, pPICK9K based recombinant expression construct was used (described in [2]). This construct is schematically depicted in Fig. 1. Here, production of the recombinant receptor is driven by a strong alcohol oxidase promoter (AOX1). The activity of this promoter is dependent on the carbon source in the culture medium and maximum activity can be achieved using methanol as a sole carbon source. For membrane targeting of the recombinant B2R, an α-factor
Discussion
A number of GPCRs have been expressed in different yeast systems such as S. cerevisiae, Schizosaccharomyces pombe and P. pastoris [22]. Out of these, P. pastoris is of special interest as it can be used for high density fermentation in order to produce large amounts of a given GPCR. Large-scale production of the human ETB endothelin receptor in P. pastoris by high density fermentation has been reported earlier [30]. Other GPCRs which have been successfully expressed in P. pastoris include the
Acknowledgments
We thank F. Joos for excellent technical assistance in the immunogold staining experiment. This work was supported by Sanofi-Aventis, the Fonds der Chemischen Industrie and the Max Planck Gesellschaft.
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