Exploiting recombination in single bacteria to make large phage antibody libraries.
The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.
Sblattero
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International School for Advanced Studies (SISSA), Biophysics Sector, Via Beirut 2-4, Trieste, 34014, Italy.
Bradbury
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D000906:Antibodies / Q000235:genetics* / Q000276:immunology*; D000916:Antibody Diversity / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D015321:Gene Rearrangement / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011995:Recombination, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014764:Viral Proteins
D000906:Antibodies; D000941:Antigens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases