title: A method to confer Protein L binding ability to any antibody fragment.
abstract: Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.
authors.affiliation: a Université de Tours, UMR1282 Infectiologie et Santé Publique, 37200 Tours, France, Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Santé Publique , 37380 Nouzilly , France.
authors.forename: Zineb
authors.initials: Z
authors.lastname: Lakhrif
authors.affiliation: b IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM Institut Régional du Cancer , Montpellier , 34090 , France.
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.affiliation: b IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM Institut Régional du Cancer , Montpellier , 34090 , France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Henriquet
authors.affiliation: a Université de Tours, UMR1282 Infectiologie et Santé Publique, 37200 Tours, France, Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Santé Publique , 37380 Nouzilly , France.
authors.forename: Anne
authors.initials: A
authors.lastname: di Tommaso
authors.affiliation: a Université de Tours, UMR1282 Infectiologie et Santé Publique, 37200 Tours, France, Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Santé Publique , 37380 Nouzilly , France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Dimier-Poisson
authors.affiliation: c Muséum National d'Histoire Naturelle, UMR MNHN-CNRS 7245, 12 rue Buffon , Paris , 75231 , France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Billiald
authors.affiliation: a Université de Tours, UMR1282 Infectiologie et Santé Publique, 37200 Tours, France, Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Santé Publique , 37380 Nouzilly , France.
authors.forename: Matthieu O
authors.initials: MO
authors.lastname: Juste
authors.affiliation: a Université de Tours, UMR1282 Infectiologie et Santé Publique, 37200 Tours, France, Institut National de la Recherche Agronomique, UMR1282 Infectiologie et Santé Publique , 37380 Nouzilly , France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Aubrey
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020125:Mutation, Missense; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: Atffinity; Fab; antibody; detection; protein L (PpL); purification; scFv