PMID: 11342716May 9, 2001Paper

Selection of novel ligands from a whole-molecule randomly mutated C5a library

Protein Engineering
S A CainP N Monk

Abstract

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.

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Oct 24, 2000·Journal of Immunological Methods·S A CainP N Monk

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Citations

Oct 5, 2007·Molecular Biotechnology·Hiroshi IshikawaRyoichi Sato
Oct 18, 2003·Biochemical Pharmacology·Stuart A CainPeter N Monk
May 15, 2003·Nuclear Medicine and Biology·H J RennenO C Boerman
Jan 22, 2005·The Journal of Biological Chemistry·Adrian HigginbottomPeter N Monk

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