Selectable in-vivo recombination to increase antibody library size - an improved phage display vector system

Phage display technology permits the display of libraries of random combina tions of light (LC) and heavy chain (HC) antibody genes. Maximizing the siz e of these libraries would enable the isolation of antibodies with high aff inity and specificity. In this study, the loxP/Cre system of in-vivo recomb ination has been employed to construct an improved vector system for the di splay of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gen e is 'silent' before recombination but active after recombination. A second acceptor phagemid, pMOX, is used for cloning the LC repertoire. Following infection with a Cre producing phage, pMOX accepts the CAT/HC library from pUX via site-specific recombination at the loxP sites. Recombinants can the n be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4 x 10(9) recombinants. Restriction analysis an d Fab expression confirmed that 100% of the colonies in the library were re combinants. This system provides a stable selectable mechanism for the gene ration of large libraries and avoids the isolation of non-recombinants enco untered with earlier in-vivo recombination systems. (C) 1999 Elsevier Scien ce B.V. All rights reserved.