TY  - JOUR
AU  - Paoletti, F.
AU  - Malerba, F.
AU  - Konarev, P. V.
AU  - Visintin, M.
AU  - Scardigli, R.
AU  - Fasulo, L.
AU  - Lamba, D.
AU  - Svergun, D. I.
AU  - Cattaneo, A.
AU  - DESY
TI  - Direct intracellular selection and biochemical characterization of a recombinant anti-proNGF single chain antibody fragment
JO  - Archives of biochemistry and biophysics
VL  - 522
SN  - 0003-9861
CY  - San Diego, Calif.
PB  - Elsevier
M1  - PHPPUBDB-24943
SP  - 26-36
PY  - 2012
AB  - 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.
KW  - Animals
KW  - Antibodies, Monoclonal: immunology
KW  - Antibodies, Monoclonal: metabolism
KW  - Antibody Affinity
KW  - Base Sequence
KW  - Circular Dichroism
KW  - Cross Reactions
KW  - DNA Primers
KW  - Enzyme-Linked Immunosorbent Assay
KW  - Humans
KW  - Isoelectric Focusing
KW  - Mice
KW  - Nerve Growth Factor: immunology
KW  - Protein Precursors: immunology
KW  - Recombinant Proteins: metabolism
KW  - Scattering, Small Angle
KW  - Single-Chain Antibodies: immunology
KW  - Single-Chain Antibodies: metabolism
KW  - Spectrometry, Fluorescence
KW  - Surface Plasmon Resonance
KW  - X-Ray Diffraction
KW  - Antibodies, Monoclonal (NLM Chemicals)
KW  - DNA Primers (NLM Chemicals)
KW  - Protein Precursors (NLM Chemicals)
KW  - Recombinant Proteins (NLM Chemicals)
KW  - Single-Chain Antibodies (NLM Chemicals)
KW  - pro-nerve growth factor, mouse (NLM Chemicals)
KW  - Nerve Growth Factor (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:22516657
UR  - <Go to ISI:>//WOS:000304789000004
DO  - DOI:10.1016/j.abb.2012.04.003
UR  - https://bib-pubdb1.desy.de/record/141215
ER  -