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 C5adR74 was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP {dP: 6-(2-deoxy-β-D-ribofu-ranosyl)-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.
| Original language | English |
|---|---|
| Pages (from-to) | 189-193 |
| Number of pages | 4 |
| Journal | Protein engineering |
| Volume | 14 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2001 |
Keywords
- C5a
- Phage display
- Polymerase chain reaction
- Pyrimidine analogue
- Random mutagenesis