Growth signalobody selects functional intrabodies in the mammalian cytoplasm

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 antig...

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Published inBiotechnology journal Vol. 11; no. 4; p. 565
Main Authors Lee, Songhee, Kaku, Yoshihiro, Inoue, Satoshi, Nagamune, Teruyuki, Kawahara, Masahiro
Format Journal Article
LanguageEnglish
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
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Summary: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.
ISSN:1860-7314
DOI:10.1002/biot.201500364