Generating molecular diversity by homologous recombination in Escherichia coli.
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.
Centre for Protein Engineering, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK. plwang@stanford.edu
Peter L
PL
Wang
Benny K C
BK
Lo
Greg
G
Winter
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002645:Chickens; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D019151:Peptide Library; D011995:Recombination, Genetic; D001618:beta-Lactamases / Q000235:genetics
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D005090:Exodeoxyribonucleases; C038225:recE protein, E coli; D015246:Deoxyribonuclease EcoRI; D001618:beta-Lactamases