title: Exploiting recombination in single bacteria to make large phage antibody libraries.
abstract: 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.
authors.affiliation: International School for Advanced Studies (SISSA), Biophysics Sector, Via Beirut 2-4, Trieste, 34014, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Sblattero
authors.forename: A
authors.initials: A
authors.lastname: Bradbury
mesh_terms: 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
chemical_list: D000906:Antibodies; D000941:Antigens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases