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

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 3.5pmol/mg of total membrane protein. [3H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (K d =0.87±0.1nM), 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. [Copyright &y& Elsevier]