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 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.
Sloan
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DD
Affymax Research Institute, Palo Alto, California 94304, USA.
Barrett
R W
RW
Tate
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EH
England
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BP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D015936:Complement C5a / Q000378:metabolism*; D006224:Cricetinae; D003902:Detergents; D004353:Drug Evaluation, Preclinical; D000939:Epitopes; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D017433:Protein Structure, Secondary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D003902:Detergents; D000939:Epitopes; D019151:Peptide Library; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a