Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris

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Abstract

The human bradykinin subtype 2 receptor (B2R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5 pmol/mg of total membrane protein. [3H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.87 ± 0.1 nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B2R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B2R suggested that the recombinant receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the recombinant receptor for structural studies.

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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