Growth signalobody selects functional intrabodies in the mammalian cytoplasm

• Published in: Biotechnology journal Vol. 11; no. 4; p. 565
• Main Authors: Lee, Songhee, Kaku, Yoshihiro, Inoue, Satoshi, Nagamune, Teruyuki, Kawahara, Masahiro
• Format: Journal Article
• Language: English
• Published: Germany 01.04.2016
• Subjects: Animals; Antigens - metabolism; Cell Line; Cell Proliferation; Cytoplasm - metabolism; Humans; Interleukin-3 - pharmacology; Mice; Peptide Library; Protein Engineering - methods; Protein Multimerization; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Signal Transduction; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism; Receptor tyrosine kinase; Library selection; Mammalian cell; Intrabody; Growth signalobody
• DOI: 10.1002/biot.201500364

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.