Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries

• Published in: Journal of immunological methods 2014-05, Vol.407, p.26-34
• Main Authors: Shahsavarian, Melody A., Le Minoux, Damien, Matti, Kalyankumar M., Kaveri, Srini, Lacroix-Desmazes, Sébastien, Boquet, Didier, Friboulet, Alain, Avalle, Bérangère, Padiolleau-Lefèvre, Séverine
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophages - genetics; Cell Surface Display Techniques; Cloning, Molecular; DNA Restriction Enzymes - genetics; DNA Restriction Enzymes - metabolism; Genetic Vectors - genetics; Immune repertoire; Mice; Mice, Inbred Strains; Nucleic Acid Amplification Techniques - methods; Phage display; Rolling circle amplification; Single chain fragment variable; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2014.03.015

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~108 clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library. •A new strategy to construct large scFv phage display libraries is described.•RCA is used to amplify scFv fragments after assembly PCR.•The efficiency of restriction enzyme digestion is significantly improved.•Library size and success of transformation are significantly increased.•We have produced a diverse phage display scFv library of size ~108.