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

Shukla, A. K., Haase, W., Reinhart, C., & Michel, H. (2007). Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 55(1), 1-8. doi:10.1016/j.pep.2007.02.021.

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Item Permalink: https://hdl.handle.net/11858/00-001M-0000-0024-D8E7-A Version Permalink: https://hdl.handle.net/21.11116/0000-000B-A21D-E
Genre: Journal Article

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 Creators:
Shukla, Arun Kumar1, Author           
Haase, Winfried2, Author           
Reinhart, Christoph1, Author           
Michel, Hartmut1, Author                 
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              

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Free keywords: GPCR; Bradykinin receptor; Heterologous expression; Glycosylation; Localization
 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.

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Language(s): eng - English
 Dates: 2007-02-142006-12-062007-03-242007-09
 Publication Status: Issued
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.pep.2007.02.021
 Degree: -

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Title: Protein Expression and Purification
Source Genre: Journal
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Publ. Info: San Diego : Academic Press
Pages: - Volume / Issue: 55 (1) Sequence Number: - Start / End Page: 1 - 8 Identifier: ISSN: 1046-5928
CoNE: https://pure.mpg.de/cone/journals/resource/954922650158