title: Growth signalobody selects functional intrabodies in the mammalian cytoplasm.
abstract: A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo-oligomeric antigen is expressed together with a single-chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin-3 (IL-3)-dependent cells. As the tyrosine kinase is activated by dimerization, only scFv-RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL-3-deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV-P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre-selected anti-RV-P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Songhee
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kaku
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inoue
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. kawahara@bio.t.u-tokyo.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawahara
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D000941:Antigens; D007377:Interleukin-3; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D011505:Protein-Tyrosine Kinases
keywords: Growth signalobody; Intrabody; Library selection; Mammalian cell; Receptor tyrosine kinase