title: Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.
abstract: 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 ~10(8) 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.
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France.
authors.forename: Melody A
authors.initials: MA
authors.lastname: Shahsavarian
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Le Minoux
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France.
authors.forename: Kalyankumar M
authors.initials: KM
authors.lastname: Matti
authors.affiliation: Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris6, UMR S 872, Paris F-75006, France; Université Paris Descartes, UMR 872, Paris F-75006, France; INSERM, UMR 872, Paris F-75006, France; International Associated Laboratory IMPACT, Institut National de la Santé et de la Recherche Médicale-France and Indian Council of Medical Research-India, National Institute of Immunohaemotology, Mumbai, India.
authors.forename: Srini
authors.initials: S
authors.lastname: Kaveri
authors.affiliation: Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris6, UMR S 872, Paris F-75006, France; Université Paris Descartes, UMR 872, Paris F-75006, France; INSERM, UMR 872, Paris F-75006, France; International Associated Laboratory IMPACT, Institut National de la Santé et de la Recherche Médicale-France and Indian Council of Medical Research-India, National Institute of Immunohaemotology, Mumbai, India.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lacroix-Desmazes
authors.affiliation: Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), iBiTecS, SPI, Commissariat à l'Energie Atomique, 91191 Gif sur Yvette, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Friboulet
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France.
authors.forename: Bérangère
authors.initials: B
authors.lastname: Avalle
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, CS 60319, 60203 Compiègne, France. Electronic address: severine.padiolleau@utc.fr.
authors.forename: Séverine
authors.initials: S
authors.lastname: Padiolleau-Lefèvre
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D057127:Single-Chain Antibodies; D004262:DNA Restriction Enzymes
keywords: Immune repertoire; Phage display; Rolling circle amplification; Single chain fragment variable