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 combinations of light (LC) and heavy chain (HC) antibody genes. Maximizing the size of these libraries would enable the isolation of antibodies with high affinity and specificity. In this study, the loxP/Cre system of in-vivo recombination has been employed to construct an improved vector system for the display of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gene 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 then be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4x109 recombinants. Restriction analysis and Fab expression confirmed that 100% of the colonies in the library were recombinants. This system provides a stable selectable mechanism for the generation of large libraries and avoids the isolation of non-recombinants encountered with earlier in-vivo recombination systems.