Selection of novel ligands from a whole-molecule randomly mutated C5a library.
Novel antagonists of the proinflammatory leukocyte chemoattractant C5a have been produced from a phage display library of whole-molecule random mutants. The cDNA for the inflammatory polypeptide C5adR(74) was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP [dP: 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one] and 8-oxodGTP (8-oxodG: 8-oxo-2'-deoxyguanosine) to allow the introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on bacteriophage M13 using a jun/fos linker sequence. Functional polypeptides were isolated by several rounds of selection against the receptor for C5a expressed on the surface of CHO cells. From this selection procedure, a limited number of variants of C5adR(74) were obtained. When expressed as free polypeptide, the binding affinities of the selected C5adR(74) sequences were increased 5-fold relative to wild-type protein. Site-directed mutagenesis of the C-terminus of these variants resulted in the production of antagonists of C5adR(74) activity.
Cain
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Krebs Institute of Biomolecular Science, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK.
Williams
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Harris
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Monk
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D015936:Complement C5a / Q000737:chemistry* / Q000235:genetics; D006224:Cricetinae; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008024:Ligands; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011951:Receptors, Complement / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D015703:Antigens, CD; D008024:Ligands; D009711:Nucleotides; D011506:Proteins; D044087:Receptor, Anaphylatoxin C5a; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015936:Complement C5a