横川 隆志 (Takashi YOKOGAWA)
専門：Genetic engineering, protein engineering, RNA engineering
略歴：
昭和62年

3月

東京大学工学部工業化学科

昭和62年

4月

東京大学大学院工学系研究科合成化学専攻修士課程

平成元年

3月

平成元年

4月

平成4年

3月

平成4年

4月

同

卒業

上

東京工業大学大学院総合理工学研究科生命化学専攻博士課程
同

上

入学
修了
入学
修了

東京工業大学助手（生命理工学部）（この間，現職のまま平成6年7月から平成7年11月までドイツ連邦共和国バイロイト大学で特別研究員）

平成7年

12月

岐阜大学講師（工学部）

平成15年

4月

岐阜大学助教授（工学部）

平成19年

4月

岐阜大学准教授（工学部）

平成25年

4月

岐阜大学教授（工学部）

平成28年

5月

岐阜大学大学院連合創薬医療情報研究科・教授を兼務

平成28年

10月

岐阜大学研究推進・社会連携機構生命の鎖統合研究センターを兼務

研究課題：（代表的な研究）
1. Study on the synthesis of super proteins carrying unnatural amino acids inserted sitespecifically
For site-specific insertion of unnatural amino acids into proteins, mutant tRNAs and mutant
aminoacyl-tRNA synthetases—artificially introduced into the protein synthesis system—should notinterfere with endogenous tRNAs or aminoacyl-tRNA synthetases. It was previously confirmed thattyrosine-tRNA and tyrosyl-tRNA synthetase from yeasts and methane-producing archaea can beintroduced into Escherichia coli without interfering with the endogenous protein synthesis system.
We have successfully created genetically modified tyrosyl-tRNA synthetase, which, unlike its wildtype counterpart, can recognize 3-azidotyrosine from yeasts and methane-producing archaea. Inorder to introduce unnatural amino acids in a site-specific manner, we also developed an efficientmethod for the preparation of an artificial amber suppressor tyrosine-tRNA, which can recognize
amber (UAG) codons that usually serve as stop codons. Furthermore, by combining both, we havealready succeeded in the site-specific introduction of 3-azidotyrosine, an unnatural amino acid, intoproteins. Because the azide group is not a constituent functional group of natural polymers,
chemical modification of introduced azide groups will add novel value to proteins. We have alreadyachieved the following: modification with biotin or fluorescence residues without affecting theactivities of the original protein; analysis of structural changes; and analysis of protein-proteininteraction. Our preparation method for proteins carrying unnatural amino acids is a patented
technology and will contribute to a number of collaborative studies.
2. Study of the structure-function relationship of modified nucleosides present in tRNAs
tRNAs play an important role in the conversion of the base sequences of genes into amino acidsequences of the corresponding proteins. One of characteristics of tRNA is the high content ofmodified nucleosides. Modified nucleosides appear to enhance the accuracy of codon recognition
and the stability of 3D structure, thereby contributing to the fine-tuning of tRNA function. Against
this background, we have been analyzing structures of modified nucleosides, and consequentchanges in tRNA function. First, we found that addition of tetraalkylammonium salt increases thetRNA-oligo DNA hybridization efficiency and we utilized this to establish a unique tRNA
purification method. This efficient tRNA purification method successfully enabled purification of
specific tRNAs and analysis of modified nucleosides that are contained within. For example, we
discovered a modified nucleoside 5-formylcytidine in bovine mitochondrial methionine-tRNA andshowed its involvement in the recognition of a non-universal genetic code (AUA for methionine
instead of isoleucine) in mitochondria. We also discovered a novel modified nucleoside,
agmatidine, in archaeal isoleucine-tRNA and found that it is essential in the recognition of the AUA
codon. We are currently studying the mechanism involved in biosynthesis of archaeosine, anarchaeal tRNA-specific modified nucleoside.
3. Study on the archaeal protein synthesis system
Archaea, lacking a nucleus, are classified as prokaryotes, but the characteristics of constituent
factors of the protein synthesis system are more similar to those of eukaryotic cells than those ofbacteria. Furthermore, by taking into consideration the number of constituent genes, the archaeal
system has the appearance of a simplified system of the complex protein synthesis system ineukaryotic cells. However, all archaea are currently culturable only under extreme conditions(hyperthermophilic, halophilic, or acidophilic), and this has been an obstacle to investigating theprotein synthesis system. We have selected several archaea that might grow at the temperaturesimilar to that used for E. coli culture. Among them, Methanosarcina acetivorans, a methaneproducing archaeon, is now culturable at the liter-scale. We are currently trying to establish a cellfree protein synthesis system and genetic engineering of this organism, and will study other archaea
to establish the archaeal protein expression system.
研究業績：（過去５年の論文）
1. Yamashita, S., Shibata, N., Boku-Ikeda, A., Abe, E., Inayama, A., Yamaguchi, T., Higuma, A.,
Inagaki, K., Tsuyuzaki, T., Iwamoto, S., Ohno, S., Yokogawa, T., Nishikawa, K., Biswas, K.B.,
Nabi, A.H., Nakagawa, T., Suzuki, F., Ebihara, A.
Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization
as a renin substrate. BMC Biotechnol., 16, 33 (2016)
2. Kawamura, T., Hirata, A., Ohno, S., Nomura, Y., Nagano, T., Nameki, N., Yokogawa, T., Hori,H.
Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasmaacidophilum, a thermo-acidophilic archaeon. Nucleic Acids Res., 44, 1894-1908 (2016)
3. Nomura, Y., Ohno, S., Nishikawa, K., Yokogawa, T.
Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of atRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase. Genes Cells, 21, 41-52(2016)
4. Inada, N., Nakamoto, K., Yokogawa, T., Ueno, Y.
Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends
and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.
Eur. J. Med. Chem., 103, 460-472 (2015)
5. Kazayama, A., Yamagami, R., Yokogawa, T., Hori, H.
Improved solid-phase DNA probe method for tRNA purification: large-scale preparation andalteration of DNA fixation. J. Biochem., 157, 411-418 (2015)
6. Ikeda-Boku, A., Kondo, K., Ohno, S., Yoshida, E., Yokogawa, T., Hayashi, N., Nishikawa, K.
Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized
via an azido group. J. Biochem., 154, 159-165 (2013)
7. Yamamoto, H., Okada, R., Iguchi, K., Ohno, S., Yokogawa, T., Nishikawa, K., Unno, K.,
Hoshino, M. and Takeda, A.

Involvement of plasmin-mediated extracellular activation of progalanin in angiogenesis.
Biochem. Biophys. Res. Commun., 430, 999-1004 (2013)
8. Ikeda-Boku, A., Ohno, S., Hibino, Y., Yokogawa, T., Hayashi, N., Nishikawa, K.
Site-selective post-translational modification of proteins using an unnatural amino acid, 3azidotyrosine. J. Biochem., 153, 317-326 (2013)
9. Nomura, Y., Onda, Y., Ohno, S., Taniguchi, H., Ando, K., Oka, N., Nishikawa, K., Yokogawa, T.
Purification and comparison of native and recombinant tRNA-guanine transglycosylases from
Methanosarcina acetivorans. Protein Expr. Purif., 88, 13-19 (2013)
10. Yoshimura, S.H., Khan, S., Ohno, S., Yokogawa, T., Nishikawa, K., Hosoya, T., Maruyama, H.,Nakayama, Y., Takeyasu, K.
Site-specific attachment of a protein to a carbon nanotube end without loss of protein function.
Bioconjug. Chem., 23, 1488-1493 (2012)
外部資金：（過去５年）
平成 23〜25 年度文部科学省 (基盤研究C) 課題番号23570208「tRNA スプライシング機構の特徴点抽出」
平成 24 年度岐阜大学研究活性化経費グループ研究支援「機能未知タンパク質の機能解明ルーティンの確立」

特許：１） 特許名：非天然蛋白質の製造方法、固定化方法及びキット
出願人：国立大学法人岐阜大学
発明者：西川一八、鈴木正昭、横川隆志、細谷孝充、大野敏
番号等：特願2003̶5747（2003年3月4日出願）

特許第3896460号（2007年1月5日登録）

２） 特許名：部位特異的にタンパク質にチロシンアナログを導入する方法
出願人：国立大学法人岐阜大学
発明者：西川一八、横川隆志、大野敏
番号等：特願2007­521381（2006年6月14日出願）

PCT/JP2006/312370
特許第4654449号（2011年1月7日登録）

３） 特許名：Ｎ末端アミノ酸が標識されたタンパク質の効率的な合成方法
出願人：バイオコゥマー株式会社
発明者：金森崇、西川一八、横川隆志、大野敏
番号等：特願2007̶545346（2006年11月16日出願）

PCT/JP2006/323378
特許第5049136号（2012年7月27日登録）

４） 特許名：タンパク質のＮ末を酵素的に修飾する方法
出願人：国立大学法人岐阜大学
発明者：西川一八、横川隆志、大野敏
番号等：特願2007̶546539（2006年11月22日出願）

PCT/JP2006/323879
特許第5061351号（2012年8月17日登録）