title: Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives.
abstract: ScFv antibody fragments are a promising alternative to full-length antibodies for both therapeutic and diagnosis applications. They can be overexpressed in bacteria, which enables easy large scale production. Since scFv are artificial constructs, they are poorly soluble and prone to aggregation, which makes them difficult to manipulate and to refold. Here, stabilization and refolding of scFv fragments from urea-unfolded solutions are reported based on the use of micromolar amounts of polymers playing the role of artificial chaperons. Using fluorescence correlation spectroscopy, the size and aggregation number of complexes of scFv with unmodified or hydrophobically modified poly(sodium acrylate) are determined. The evolution of the secondary structure along the refolding procedure, in the presence or absence of 0.4 m l-arginine at scFv:polymer < 1:5 (w/w), is determined by high-sensitivity synchrotron-radiation circular dichroism. Measurements reveal that refolding in the presence of polymers yields native-like secondary structure, though a different folding pathway can be followed compared to refolding in the absence of polymer. This is the first report on the use of macromolecular additives to assist refolding of a multidomain protein of therapeutic interest.
authors.affiliation: Ecole normale supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Département de Chimie, PASTEUR, 24, rue Lhomond, 75005, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, ENS, CNRS, PASTEUR, 75005, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Martin
authors.affiliation: CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), Bt. 136, CEA Saclay, F-91191, Gif sur Yvette, France.
authors.forename: Narciso
authors.initials: N
authors.lastname: Costa
authors.affiliation: Synchrotron Soleil, Saint-Aubin, F-91192, Gif-sur-Yvette, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Wien
authors.affiliation: Department of Chemistry, Faculty of Pharmacy, Université de Montréal, CP 6128 Succursale Centre Ville, Montréal, QC, H3C 3J7, Canada.|World Premier Initiative (WPI) International Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA) and National Institute for Materials Science (NIMS) 1-1Namiki, Tsukuba, 305-0044, Japan.|Department of Chemistry and Faculty of Pharmacy, University of Helsinki, Helsinki, FI, 00014, Finland.
authors.forename: Françoise M
authors.initials: FM
authors.lastname: Winnik
authors.affiliation: CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), Bt. 136, CEA Saclay, F-91191, Gif sur Yvette, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Ortega
authors.affiliation: CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), Bt. 136, CEA Saclay, F-91191, Gif sur Yvette, France.
authors.forename: Amaury
authors.initials: A
authors.lastname: Herbet
authors.affiliation: CEA, iBiTecS, SPI, Laboratoire d'Ingénierie des Anticorps pour la Santé (LIAS), Bt. 136, CEA Saclay, F-91191, Gif sur Yvette, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
authors.affiliation: Ecole normale supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Département de Chimie, PASTEUR, 24, rue Lhomond, 75005, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, ENS, CNRS, PASTEUR, 75005, Paris, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Tribet
mesh_terms: D000180:Acrylic Resins / Q000138:chemical synthesis / Q000737:chemistry*; D002942:Circular Dichroism; D003956:Dialysis; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D057446:Hydrodynamics; D057927:Hydrophobic and Hydrophilic Interactions; D066329:Protein Aggregates; D017510:Protein Folding; D017433:Protein Structure, Secondary; D057127:Single-Chain Antibodies / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
chemical_list: D000180:Acrylic Resins; D066329:Protein Aggregates; D057127:Single-Chain Antibodies; C006903:carbopol 940; D014508:Urea; D019791:Guanidine
keywords: antibodies; chaperon-like polymers; fluorescence correlation spectroscopy; protein folding; synchrotron-radiation circular dichroism