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Growth signalobody selects functional intrabodies in the mammalian cytoplasm
Author(s) -
Lee Songhee,
Kaku Yoshihiro,
Inoue Satoshi,
Nagamune Teruyuki,
Kawahara Masahiro
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201500364
Subject(s) - cytoplasm , biology , phosphoprotein , microbiology and biotechnology , tyrosine kinase , antigen , signal transduction , phosphorylation , genetics
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.