Expression of an Epitope Tagged Human C5a Receptor and Antibody-Mediated Immobilization of Detergent-Solubilized Receptor for Drug Discovery Screening

In order to screen combinatorial libraries of peptides and/or small organic molecules against the human C5a receptor, we have developed a novel method for immobilizing and screening 7-transmembrane segment (7-TMS) receptors. The epitope for a high affinity monoclonal antibody (mAb179) was added to t...

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Bibliographic Details
Published in:Protein expression and purification 1997-10, Vol.11 (1), p.119-124
Main Authors: Sloan, Derek D., Barrett, Ronald W., Tate, Emily H., England, Bruce P.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal
Antigens, CD - biosynthesis
Antigens, CD - chemistry
Antigens, CD - metabolism
CHO Cells
Complement C5a - metabolism
Cricetinae
Detergents
Drug Evaluation, Preclinical
Epitopes
Humans
Molecular Sequence Data
Peptide Library
Protein Structure, Secondary
Receptor, Anaphylatoxin C5a
Receptors, Complement - biosynthesis
Receptors, Complement - chemistry
Receptors, Complement - metabolism
Solubility
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Summary:In order to screen combinatorial libraries of peptides and/or small organic molecules against the human C5a receptor, we have developed a novel method for immobilizing and screening 7-transmembrane segment (7-TMS) receptors. The epitope for a high affinity monoclonal antibody (mAb179) was added to the C-terminus of the human C5a receptor, and CHO cell lines expressing the epitope tagged receptor (C5aR-KH) and the wild-type receptor (C5aR) were established. The addition of the epitope tag did not affect the affinity of C5aR-KH for C5a. The epitope tag allowed for mAb179-mediated immobilization of active receptor either in intact membranes or following detergent extraction with digitonin or Chaps. If this method is generally applicable to other 7-TMS receptors it may have broad utility for drug discovery screening as well as other applications.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1997.0777