title: Direct intracellular selection and biochemical characterization of a recombinant anti-proNGF single chain antibody fragment.
abstract: proNGF, the precursor of the neurotrophin NGF, is widely expressed in central and peripheral nervous system. Its physiological functions are still largely unknown, although it emerged from studies in the last decade that proNGF has additional and distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. The regulation of proNGF/NGF ratio represents a crucial process for homeostasis of brain and other tissues, and understanding the molecular aspects of these differences is important. We report the selection and characterization of a recombinant monoclonal anti-proNGF antibody in single chain Fv fragment (scFv) format. The selection exploited the Intracellular Antibody Capture Technology (IACT), starting from a naïve mouse SPLINT (Single Pot Library of INTracellular antibodies) library. This antibody (scFv FPro10) was expressed recombinantly in Escherichia coli, was proven to be highly soluble and stable, and thoroughly characterized from the biochemical-biophysical point of view. scFv FPro10 displays high affinity and specificity for proNGF, showing no cross-reactivity with other pro-neurotrophins. A structural model was obtained by SAXS. scFv FPro10 represents a new tool to be exploited for the selective immunoanalysis of proNGF, both in vitro and in vivo, and might help in understanding the molecular function of proNGF in neurodegeneration.
authors.affiliation: EBRI-European Brain Research Institute, Via del Fosso di Fiorano 64, 00143 Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Paoletti
authors.forename: Francesca
authors.initials: F
authors.lastname: Malerba
authors.forename: Petr V
authors.initials: PV
authors.lastname: Konarev
authors.forename: Michela
authors.initials: M
authors.lastname: Visintin
authors.forename: Raffaella
authors.initials: R
authors.lastname: Scardigli
authors.forename: Luisa
authors.initials: L
authors.lastname: Fasulo
authors.forename: Doriano
authors.initials: D
authors.lastname: Lamba
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Antonino
authors.initials: A
authors.lastname: Cattaneo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D002942:Circular Dichroism; D003429:Cross Reactions; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007525:Isoelectric Focusing; D051379:Mice; D020932:Nerve Growth Factor / Q000276:immunology*; D011498:Protein Precursors / Q000276:immunology*; D011994:Recombinant Proteins / Q000378:metabolism; D053838:Scattering, Small Angle; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014961:X-Ray Diffraction
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011498:Protein Precursors; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C489226:pro-nerve growth factor, mouse; D020932:Nerve Growth Factor