Engineering a growth sensor to select intracellular antibodies in the cytosol of mammalian cells

• Published in: Journal of bioscience and bioengineering Vol. 124; no. 1; pp. 125 - 132
• Main Authors: Nguyen, Thuy Duong, Takasuka, Hitoshi, Kaku, Yoshihiro, Inoue, Satoshi, Nagamune, Teruyuki, Kawahara, Masahiro
• Format: Journal Article
• Language: English
• Published: Japan Elsevier B.V 01.07.2017
• Subjects: Animals; antibodies; Antibodies - genetics; Antibodies - immunology; antigens; bacteriophages; c-kit; cytosol; Cytosol - metabolism; engineering; Growth signal; Intracellular antibody; Library screening; Mammalian cell; mammals; nucleoproteins; Nucleoproteins - immunology; Peptide Library; Protein Engineering - methods; Rabies lyssavirus; Rabies virus; Receptor engineering; therapeutics; Library screening; Receptor engineering; Mammalian cell; c-kit; Intracellular antibody; Growth signal; Rabies virus
• ISSN: 1389-1723; 1347-4421; 1347-4421
• DOI: 10.1016/j.jbiosc.2017.02.017

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.