Journal of Immunological Methods 245 (2000) 139–145 www.elsevier.nl / locate / jim Recombinant Technology Analysis of receptor / ligand interactions using whole-molecule randomly-mutated ligand libraries a a,c b a,b Stuart A. Cain , Charlotte F. Ratcliffe , David M. Williams , Victoria Harris , a, * Peter N. Monk a Krebs Institute of Biomolecular Science, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK b Department of Chemistry, University of Sheffield, Sheffield S10 2UH, UK c Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195, USA Received 20 April 2000; received in revised form 3 August 2000; accepted 3 August 2000 Abstract We report a novel method for the analysis of protein ligands using a whole molecule mutagenesis/phage display system. The cDNA for the inflammatory polypeptide C5a was used as template in a PCR reaction doped with mutagenic nucleoside triphosphates (dP and 8-oxo-29deoxyguanosine (8-oxodG)) to allow introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on the surface of phage and functional polypeptides were selected by several rounds of selection against the cells bearing the receptor for C5a. Following selection only a limited number of residues in C5a were found to be mutated, suggesting that mutations in key residues involved in the maintenance of structure and in receptor binding had been eliminated. The selected C5a sequences had a higher affinity for receptor than wild type phage-C5a conjugates. As this method for analysing the functional characteristics of proteins does not rely on knowledge a priori of structure, it may be useful for affinity maturation or analysis in a wide range of protein ligand / receptor systems.  2000 Elsevier Science B.V. All rights reserved. Keywords: C5a; Random mutagenesis; Phage display; Pyrimidine analogue; PCR; 8-oxodeoxyguanosine 1. Introduction Abbreviations: dP, 6-(2-deoxy-b-D-ribofuranosyl)-3,4-dihydro- 8H-pyrimido-[4,5-C][1,2]oxazin-7-one; 8-oxodG, 8-oxo-29deox- The binding of polypeptide ligands to cell surface yguanosine; C5aR, receptor for complement fragment 5a; 74 C5adR , metabolite of C5a lacking the C-terminal arginine receptors is a summation of the individual contact residue; f-C5a, Fos-human C5a attached to phage coat protein sites that confer affinity and specificity. Conventional III-Jun; DWB, DMEM medium, 1% (w/v) BSA, 20 mM HEPES analysis of ligand / receptor interaction involves the pH 7.2; rhC5a, recombinant human C5a mutation of individual amino acids, chosen perhaps *Corresponding author. Tel.: 144-114-2224-2334; fax: 144- from knowledge of the three-dimensional structure or 114-272-8697. E-mail address: p.monk@shef.ac.uk (P.N. Monk). cross-species conservation, followed by measure- 0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759(00)00282-9