PMID:15983006
| Citation |
Wang, PL, Lo, BK and Winter, G (2005) Generating molecular diversity by homologous recombination in Escherichia coli. Protein Eng. Des. Sel. 18:397-404 |
|---|---|
| Abstract |
We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range. |
| Links |
PubMed Online version:10.1093/protein/gzi042 |
| Keywords |
Animals; Bacteriophage lambda/genetics; Chickens; DNA Damage; DNA, Bacterial/genetics; Deoxyribonuclease EcoRI/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Exodeoxyribonucleases/genetics; Exodeoxyribonucleases/metabolism; Immunoglobulin Variable Region/biosynthesis; Immunoglobulin Variable Region/genetics; Peptide Library; Recombination, Genetic; beta-Lactamases/genetics |
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