Engineering a growth sensor to select intracellular antibodies in the cytosol of mammalian cells.
Intracellular antibodies (intrabodies) are expected to function as therapeutics as well as tools for elucidating in vivo function of proteins. In this study, we propose a novel intrabody selection method in the cytosol of mammalian cells by utilizing a growth signal, induced by the interaction of the target antigen and an scFv-c-kit growth sensor. Here, we challenge this method to select specific intrabodies against rabies virus nucleoprotein (RV-N) for the first time. As a result, we successfully select antigen-specific intrabodies from a naïve synthetic library using phage panning followed by our growth sensor-based intracellular selection method, demonstrating the feasibility of the method. Additionally, we succeed in improving the response of the growth sensor by re-engineering the linker region of its construction. Collectively, the described selection method utilizing a growth sensor may become a highly efficient platform for selection of functional intrabodies in the future.
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Thuy Duong
TD
Nguyen
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Hitoshi
H
Takasuka
Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
Yoshihiro
Y
Kaku
Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
Satoshi
S
Inoue
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Teruyuki
T
Nagamune
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address: kawahara@bio.t.u-tokyo.ac.jp.
Masahiro
M
Kawahara
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D003600:Cytosol / Q000378:metabolism*; D009698:Nucleoproteins / Q000276:immunology; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011820:Rabies virus
D000906:Antibodies; D009698:Nucleoproteins; D019151:Peptide Library
Growth signal; Intracellular antibody; Library screening; Mammalian cell; Rabies virus; Receptor engineering; c-kit