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Protein Engineering, Vol. 14, No. 3, 189-193, March 2001
© 2001 Oxford University Press

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

S.A. Cain1,, D.M. Williams2,, V. Harris1,2, and P.N. Monk1,3,

Krebs Institute of Biomolecular Science, 1 Department of Molecular Biology and Biotechnology and 2 Department of Chemistry, University of Sheffield, Sheffield S10 2UH, UK

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 C5adR74 was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP {dP: 6-(2-deoxy-ß-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 C5adR74 were obtained. When expressed as free polypeptide, the binding affinities of the selected C5adR74 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 C5adR74 activity.


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A. Higginbottom, S. A. Cain, T. M. Woodruff, L. M. Proctor, P. K. Madala, J. D. A. Tyndall, S. M. Taylor, D. P. Fairlie, and P. N. Monk
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