High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split<i>Renilla</i>luciferase

• Sung Bae Kim

  Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; and SC BioSciences Corporation, 2-2-11 Shiba-Daimon, Minato-ku, Tokyo 105-0012, Japan

• Takeaki Ozawa

  Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; and SC BioSciences Corporation, 2-2-11 Shiba-Daimon, Minato-ku, Tokyo 105-0012, Japan

• Shigeaki Watanabe

  Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; and SC BioSciences Corporation, 2-2-11 Shiba-Daimon, Minato-ku, Tokyo 105-0012, Japan

• Yoshio Umezawa

  Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan; and SC BioSciences Corporation, 2-2-11 Shiba-Daimon, Minato-ku, Tokyo 105-0012, Japan

抄録

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo . The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

収録刊行物

• Proceedings of the National Academy of Sciences

  Proceedings of the National Academy of Sciences 101 (32), 11542-11547, 2004-08-02

  Proceedings of the National Academy of Sciences

キーワード

• Multidisciplinary

詳細情報

• CRID: 1363951794745558144
• NII論文ID: 30016241498
• DOI: 10.1073/pnas.0401722101
• ISSN: 10916490
• ISSN: 00278424
• Web Site: https://pnas.org/doi/pdf/10.1073/pnas.0401722101
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