Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris.
The human bradykinin subtype 2 receptor (B(2)R), 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. [(3)H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (K(d)=0.87+/-0.1 nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B(2)R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B(2)R 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.
Shukla
Arun Kumar
AK
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany. Arun.shukla@mpibp-frankfurt.mpg.de
Haase
Winfried
W
Reinhart
Christoph
C
Michel
Hartmut
H
D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D008697:Methane / Q000378:metabolism; D010843:Pichia / Q000737:chemistry / Q000235:genetics*; D043782:Receptor, Bradykinin B2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry
D043782:Receptor, Bradykinin B2; D011994:Recombinant Proteins; D008697:Methane