Selection of novel ligands from a whole-molecule randomly mutated C5a library |
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Authors: | Cain SA; Williams DM; Harris V; Monk PN |
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Affiliation: | Krebs Institute of Biomolecular Science,
1 Department of Molecular Biology and Biotechnology and
2 Department of Chemistry, University of Sheffield, Sheffield S10 2UH, UK |
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Abstract: | Novel antagonists of the proinflammatory leukocyte chemoattractantC5a have been produced from a phage display library of whole-moleculerandom mutants. The cDNA for the inflammatory polypeptide C5adR74was used as template in a PCR reaction doped with the mutagenicnucleoside 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 theintroduction of mutations in a highly controlled manner throughoutthe cDNA. The resultant library of mutants was displayed onbacteriophage M13 using a jun/fos linker sequence. Functionalpolypeptides were isolated by several rounds of selection againstthe receptor for C5a expressed on the surface of CHO cells.From this selection procedure, a limited number of variantsof C5adR74 were obtained. When expressed as free polypeptide,the binding affinities of the selected C5adR74 sequences wereincreased 5-fold relative to wild-type protein. Site-directedmutagenesis of the C-terminus of these variants resulted inthe production of antagonists of C5adR74 activity. |
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