2023/12/20 更新

写真b

ムカイ タカヒト
向井 崇人
MUKAI Takahito
*大学が定期的に情報更新している項目(その他は、researchmapの登録情報を転載)
所属*
理学部
職名*
助教
研究キーワード
  • tRNA

  • バイオインフォマティクス

  • 非天然型アミノ酸

  • 遺伝暗号

  • 合成生物学

  • 学内職務経歴*
    • 2023年4月 - 現在 
      理学部   助教
    • 2018年4月 - 2023年3月 
      理学部   生命理学科   助教
    プロフィール
    研究テーマは,遺伝暗号の人為的改変と,自然界における特殊な遺伝暗号の探索です.生命の起源と可能性を追い求めています.
     

    研究分野

    • ライフサイエンス / ゲノム生物学

    経歴

    • 2018年4月 - 現在 
      立教大学   理学部生命理学科   助教(令和5年度からはCREST特任)

      詳細を見る

    • 2016年4月 - 2018年3月 
      Yale大学   Department of Molecular Biophysics and Biochemistry   日本学術振興会 海外特別研究員 (Yale大学においては、平成28年度中はPostdoctoral Fellowの身分で、平成29年度からはAssociate Research Scientistの身分)

      詳細を見る

    • 2014年6月 - 2016年3月 
      Yale大学   Department of Molecular Biophysics and Biochemistry   ポスドク

      詳細を見る

    • 2012年4月 - 2014年5月 
      理化学研究所   生命分子システム基盤研究領域(平成25年度から改組してライフサイエンス技術基盤研究センター)   基礎科学特別研究員

      詳細を見る

    • 2011年4月 - 2012年3月 
      理化学研究所   生命分子システム基盤研究領域   特別研究員

      詳細を見る

    • 2008年4月 - 2011年3月 
      東京大学大学院   理学系研究科生物化学専攻   日本学術振興会特別研究員DC1

      詳細を見る

    ▼全件表示

    学歴

    • 2006年4月 - 2011年3月 
      東京大学大学院   理学系研究科   生物化学専攻

      詳細を見る

    • 2002年4月 - 2006年3月 
      東京大学   理科一類、理学部生物化学科

      詳細を見る

    論文

    • Recoding UAG to selenocysteine in Saccharomyces cerevisiae 査読有り

      Kyle S Hoffman, Christina Z Chung, Takahito Mukai, Natalie Krahn, Han-Kai Jang, Nileeka Balasuriya, Patrick O'Donoghue, Dieter Söll

      RNA   rna.079658.123 - rna.079658.123   2023年6月6日

      詳細を見る

      掲載種別:研究論文(学術雑誌)   出版者・発行元:Cold Spring Harbor Laboratory  

      Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system, however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel selenocysteine biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these selenocysteine pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded selenocysteine. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of selenocysteine.

      DOI: 10.1261/rna.079658.123

      researchmap

    • Ancestral Archaea Expanded the Genetic Code with Pyrrolysine 査読有り

      Li-Tao Guo, Kazuaki Amikura, Han-Kai Jiang, Takahito Mukai, Xian Fu, Yane-Shih Wang, Patrick O’Donoghue, Dieter Söll, Jeffery M. Tharp

      Journal of Biological Chemistry   102521 - 102521   2022年9月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1016/j.jbc.2022.102521

      researchmap

    • Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation 査読有り

      Hironobu Fujita, Ayane Osaku, Yuto Sakane, Koki Yoshida, Kayoko Yamada, Seia Nara, Takahito Mukai, Masayuki Su’etsugu

      ACS Synthetic Biology   2022年8月23日

      詳細を見る

      担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1021/acssynbio.2c00353

      researchmap

    • The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism 査読有り

      Haolin Zhang, Xuemei Gong, Qianqian Zhao, Takahito Mukai, Oscar Vargas-Rodriguez, Huiming Zhang, Yuxing Zhang, Paul Wassel, Kazuaki Amikura, Julie Maupin-Furlow, Yan Ren, Xun Xu, Yuri I Wolf, Kira S Makarova, Eugene V Koonin, Yue Shen, Dieter Söll, Xian Fu

      Nucleic Acids Research   2022年4月25日

      詳細を見る

      掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press (OUP)  

      Abstract

      Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

      DOI: 10.1093/nar/gkac271

      researchmap

    • Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems 査読有り

      Takahito Mukai, Kazuaki Amikura, Xian Fu, Dieter Söll, Ana Crnković

      Frontiers in Genetics12   2022年1月3日

      詳細を見る

      担当区分:筆頭著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Frontiers Media SA  

      Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique <italic>O</italic>-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid <italic>O</italic>-phosphoserine (Sep) onto tRNA<sup>Cys</sup>. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNA<sup>Cys</sup> to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m<sup>1</sup>G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual <italic>O</italic>-phosphoseryl-tRNA<sup>Sec</sup> kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

      DOI: 10.3389/fgene.2021.794509

      researchmap

    • Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes 査読有り 国際誌

      Tatsuya Yoneji, Hironobu Fujita, Takahito Mukai, Masayuki Su’etsugu

      Nucleic Acids Research   2021年4月

      詳細を見る

      担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1093/nar/gkab298

      PubMed

      researchmap

    • Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria 招待有り 査読有り

      Takahito Mukai

      IJMS22 ( 9 ) 4605   2021年4月

      詳細を見る

      担当区分:筆頭著者, 最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

      researchmap

    • Rational Design of Aptamer-Tagged tRNAs 招待有り 査読有り

      Takahito Mukai

      IJMS21 ( 20 ) 7793   2020年10月

      詳細を見る

      担当区分:筆頭著者, 最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

      researchmap

    • Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli 査読有り

      Takahito Mukai, Tatsuya Yoneji, Kayoko Yamada, Hironobu Fujita, Seia Nara, Masayuki Su’etsugu

      ACS Synthetic Biology   2020年5月27日

      詳細を見る

      担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Chemical Society (ACS)  

      DOI: 10.1021/acssynbio.0c00008

      researchmap

    • A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation. 査読有り

      Hoffman KS, Vargas-Rodriguez O, Bak DW, Mukai T, Woodward LK, Weerapana E, Söll D, Reynolds NM

      The Journal of biological chemistry   2019年7月

    • Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins. 招待有り 査読有り 国際誌

      Adachi J, Katsura K, Seki E, Takemoto C, Shirouzu M, Terada T, Mukai T, Sakamoto K, Yokoyama S

      International journal of molecular sciences20 ( 3 )   2019年1月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.3390/ijms20030492

      PubMed

      researchmap

    • Engineering an auto-maturing transglutaminase with enhanced thermostability by genetic code expansion with two codon reassignments 査読有り

      Kazumasa Ohtake, Takahito Mukai, Fumie Iraha, Mihoko Takahashi, Ken-ichi Haruna, Masayo Date, Keiichi Yokoyama, Kensaku Sakamoto

      ACS Synthetic Biology   2018年7月

      詳細を見る

      記述言語:英語  

      DOI: 10.1021/acssynbio.8b00157

      researchmap

    • Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNACys and elongation factor SelB 招待有り 査読有り

      Oscar Vargas-Rodriguez, Markus Englert, Anna Merkuryev, Takahito Mukai, Dieter Söll

      RNA Biology   2018年6月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1080/15476286.2018.1474074

      researchmap

    • Eine einfache Methode zur Produktion von Selenoproteinen 招待有り 査読有り

      Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D

      Angewandte Chemie   2018年5月

      詳細を見る

      記述言語:ドイツ語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1002/ange.201713215

      researchmap

    • A facile method for producing selenocysteine‐containing proteins. 査読有り

      Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D

      Angewandte Chemie International Editiondoi: 10.1002/anie.201713215   2018年4月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1002/anie.201713215

      PubMed

      researchmap

    • RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea 査読有り

      Takahito Mukai, Ana Crnkovic, Takuya Umehara, Natalia N. Ivanova, Nikos C. Kyrpides, Dieter Soll

      MBIO8 ( 3 ) doi: 10.1128/mBio.00561-17   2017年5月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

      The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS.SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)-and pyrrolysine (Pyl)-utilizing traits in SepRSutilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.
      IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE-and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

      DOI: 10.1128/mBio.00561-17

      PubMed

      researchmap

    • Transfer RNAs with novel cloverleaf structures 査読有り

      Takahito Mukai, Oscar Vargas-Rodriguez, Markus Englert, H. James Tripp, Natalia N. Ivanova, Edward M. Rubin, Nikos C. Kyrpides, Dieter Soell

      NUCLEIC ACIDS RESEARCH45 ( 5 ) 2776 - 2785   2017年3月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

      We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory non-coding RNAs. In delta-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

      DOI: 10.1093/nar/gkw898

      PubMed

      researchmap

    • Bioinformatic Analysis Reveals Archaeal tRNA<sup>Tyr</sup> and tRNA<sup>Trp</sup> Identities in Bacteria. 招待有り 査読有り

      Mukai T, Reynolds NM, Crnković A, Söll D

      Life (Basel, Switzerland)7 ( 1 )   2017年2月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.3390/life7010008

      PubMed

      researchmap

    • Rewriting the Genetic Code 招待有り 査読有り

      Takahito Mukai, Marc J. Lajoie, Markus Englert, Dieter Soll

      ANNUAL REVIEW OF MICROBIOLOGY, VOL 7171 ( 1 ) 557 - 577   2017年

      詳細を見る

      記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:ANNUAL REVIEWS  

      The genetic code-the language used by cells to translate their genomes into proteins that perform many cellular functions-is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

      DOI: 10.1146/annurev-micro-090816-093247

      PubMed

      researchmap

    • Facile Recoding of Selenocysteine in Nature 査読有り

      Takahito Mukai, Markus Englert, H. James Tripp, Corwin Miller, Natalia N. Ivanova, Edward M. Rubin, Nikos C. Kyrpides, Dieter Soell

      ANGEWANDTE CHEMIE-INTERNATIONAL EDITION55 ( 17 ) 5337 - 5341   2016年4月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

      Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4trillion base pairs of metagenomic sequences and 24903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with Se-75 or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E.coli to synthesize active formate dehydrogenaseH, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

      DOI: 10.1002/anie.201511657

      PubMed

      J-GLOBAL

      researchmap

    • Leichte Neucodierung von Selenocystein in der Natur. 招待有り 査読有り

      Mukai T, Englert M, Tripp HJ, Miller C, Ivanova NN, Rubin EM, Kyrpides NC, Söll D

      Angewandte Chemie (Weinheim an der Bergstrasse, Germany)128 ( 17 ) 5423 - 5427   2016年4月

      詳細を見る

      記述言語:ドイツ語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1002/ange.201511657

      PubMed

      researchmap

    • Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli 査読有り

      Takahito Mukai, Atsushi Yamaguchi, Kazumasa Ohtake, Mihoko Takahashi, Akiko Hayashi, Fumie Iraha, Satoshi Kira, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Hiroko Hoshi, Takatsugu Kobayashi, Kensaku Sakamoto

      Nucleic Acids Research43 ( 16 ) 8111 - 8122   2015年9月18日

      詳細を見る

      担当区分:筆頭著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press (OUP)  

      DOI: 10.1093/nar/gkv787

      PubMed

      researchmap

    • Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon 査読有り

      Takahito Mukai, Hiroko Hoshi, Kazumasa Ohtake, Mihoko Takahashi, Atsushi Yamaguchi, Akiko Hayashi, Shigeyuki Yokoyama, Kensaku Sakamoto

      SCIENTIFIC REPORTS5   9699   2015年5月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

      Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.Delta A grew as vigorously as BL21(DE3) in rich medium at 25-42 degrees C, and its derivative B-95.Delta A Delta fabR was better adapted to low temperatures and minimal media than B-95.Delta A. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.Delta A strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

      DOI: 10.1038/srep09699

      PubMed

      researchmap

    • Protein stabilization utilizing a redefined codon 査読有り

      Kazumasa Ohtake, Atsushi Yamaguchi, Takahito Mukai, Hiroki Kashimura, Nobutaka Hirano, Mitsuru Haruki, Sosuke Kohashi, Kenji Yamagishi, Kazutaka Murayama, Yuri Tomabechi, Takashi Itagaki, Ryogo Akasaka, Masahito Kawazoe, Chie Takemoto, Mikako Shirouzu, Shigeyuki Yokoyama, Kensaku Sakamoto

      SCIENTIFIC REPORTS5   9762   2015年5月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

      Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-L-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

      DOI: 10.1038/srep09762

      PubMed

      researchmap

    • Multiple site-specific installations of Nε-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis. 招待有り 査読有り

      Yanagisawa T, Takahashi M, Mukai T, Sato S, Wakamori M, Shirouzu M, Sakamoto K, Umehara T, Yokoyama S

      Chembiochem : a European journal of chemical biology15 ( 12 ) 1830 - 1838   2014年8月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1002/cbic.201402291

      PubMed

      researchmap

    • Efficient Decoding of the UAG Triplet as a Full-Fledged Sense Codon Enhances the Growth of a prfA-Deficient Strain of Escherichia coli 査読有り

      Kazumasa Ohtake, Aya Sato, Takahito Mukai, Nobumasa Hino, Shigeyuki Yokoyama, Kensaku Sakamoto

      JOURNAL OF BACTERIOLOGY194 ( 10 ) 2606 - 2613   2012年5月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

      We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q(3), translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of "backup" stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.

      DOI: 10.1128/JB.00195-12

      PubMed

      researchmap

    • Wide-range protein photo-crosslinking achieved by a genetically encoded N(ε)-(benzyloxycarbonyl)lysine derivative with a diazirinyl moiety. 査読有り

      Yanagisawa T, Hino N, Iraha F, Mukai T, Sakamoto K, Yokoyama S

      Molecular bioSystems8 ( 4 ) 1131 - 1135   2012年

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)  

      DOI: 10.1039/c2mb05321g

      PubMed

      researchmap

    • Genetic-code evolution for protein synthesis with non-natural amino acids 査読有り

      Takahito Mukai, Tatsuo Yanagisawa, Kazumasa Ohtake, Masatoshi Wakamori, Jiro Adachi, Nobumasa Hino, Aya Sato, Takatsugu Kobayashi, Akiko Hayashi, Mikako Shirouzu, Takashi Umehara, Shigeyuki Yokoyama, Kensaku Sakamoto

      BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS411 ( 4 ) 757 - 761   2011年8月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites. (C) 2011 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.bbrc.2011.07.020

      PubMed

      researchmap

    • (博士論文) UAG コドン解読分子の操作による遺伝暗号可塑性の研究 査読有り

      向井崇人

      東京大学   2011年3月

      詳細を見る

      記述言語:日本語   掲載種別:学位論文(博士)  

      researchmap

    • Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells 査読有り

      Nobumasa Hino, Masaaki Oyama, Aya Sato, Takahito Mukai, Fumie Iraha, Akiko Hayashi, Hiroko Kozuka-Hata, Tadashi Yamamoto, Shigeyuki Yokoyama, Kensaku Sakamoto

      JOURNAL OF MOLECULAR BIOLOGY406 ( 2 ) 343 - 353   2011年2月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

      Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-L-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein protein interactions in cells. (C) 2010 Elsevier Ltd. All rights reserved.

      DOI: 10.1016/j.jmb.2010.12.022

      PubMed

      researchmap

    • Codon reassignment in the Escherichia coli genetic code 査読有り

      Takahito Mukai, Akiko Hayashi, Fumie Iraha, Aya Sato, Kazumasa Ohtake, Shigeyuki Yokoyama, Kensaku Sakamoto

      NUCLEIC ACIDS RESEARCH38 ( 22 ) 8188 - 8195   2010年12月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

      Most organisms, from Escherichia coli to humans, use the &apos;universal&apos; genetic code, which have been unchanged or &apos;frozen&apos; for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.

      DOI: 10.1093/nar/gkq707

      PubMed

      researchmap

    • Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells 査読有り

      Takahito Mukai, Motoaki Wakiyama, Kensaku Sakamoto, Shigeyuki Yokoyama

      PROTEIN SCIENCE19 ( 3 ) 440 - 448   2010年3月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JOHN WILEY & SONS INC  

      Insect cells are useful for the high-yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell-based system for incorporating non-natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA(Tyr) in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl-tRNA synthetase specific for 3-iodo-L-tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non-natural amino acids, and the "transient" method generated a sevenfold higher yield. By this method, 4-azido-L-phenylalanine was incorporated into human interleukin-8 at a specific site. The yield of the azido-containing IL-8 was 1 mu g/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.

      DOI: 10.1002/pro.322

      PubMed

      researchmap

    • Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases 査読有り

      Takahito Mukai, Takatsugu Kobayashi, Nobumasa Hino, Tatsuo Yanagisawa, Kensaku Sakamoto, Shigeyuki Yokoyama

      BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS371 ( 4 ) 818 - 822   2008年7月

      詳細を見る

      記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      We report a method for site-specifically incorporating L-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PyIRS)-tRNA(PYl) pair from Methanosarcino mazei. Different types of external promoters were tested for the expression of tRNA(PYl) in Chinese hamster ovary cells. When tRNA(PYl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(PYl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N-epsilon-tert-butyloxycarbonyl-L-lysine (Boc-lysine), into proteins at the amber position. This PyIRS-tRNA(PYl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PyIRS variant specific to N-epsilon-benzyloxycarbonyl-L-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N-epsilon-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells. (c) 2008 Elsevier Inc. All rights reserved.

      DOI: 10.1016/j.bbrc.2008.04.164

      PubMed

      researchmap

    ▼全件表示

    講演・口頭発表等

    • シンプル ゲノム合成 招待有り

      向井崇人

      第17回日本ゲノム微生物学会年会  2023年3月9日 

      詳細を見る

    • ゾンビ細胞のためのセントラルドグマ 招待有り

      向井崇人

      第45回日本分子生物学会年会  2022年12月2日 

      詳細を見る

      記述言語:英語   会議種別:シンポジウム・ワークショップ パネル(指名)  

      researchmap

    • Chromosome shuttling between cell-free systems and E. coli cells 招待有り

      Takahito Mukai

      The 3rd International BioDesign Research Conference  2022年11月27日 

      詳細を見る

    • ゲノムのミライ 作れる細胞の設計

      向井 崇人

      第16回日本ゲノム微生物学会年会  2022年3月2日 

      詳細を見る

      会議種別:口頭発表(一般)  

      researchmap

    • 大腸菌ゲノムの分断化とポータブル化

      向井崇人, 末次正幸

      第17回21世紀大腸菌研究会  2021年8月20日 

      詳細を見る

      会議種別:口頭発表(一般)  

      researchmap

    • 大腸菌を用いた1 Mbプラスミドの構築と接合伝達

      向井崇人, 米司達哉, 末次正幸, 藤田裕寛

      第14回日本ゲノム微生物学会年会  2020年3月8日  日本ゲノム微生物学会

      詳細を見る

      記述言語:日本語   会議種別:口頭発表(一般)  

      開催地:名古屋  

      新型コロナウイルス感染症対策の基本方針に基づき、年会を中止し、発表が行われたと認定

      researchmap

    • 地球規模メタゲノムデータの解析 SepRS、どこ行った? 招待有り

      向井 崇人

      第18回微生物研究会「微生物研究の新しい潮流」  2019年11月9日 

      詳細を見る

      記述言語:日本語  

      researchmap

    • Megabase cloning by utilizing E. coli chromosome vectors 招待有り

      向井 崇人

      ICG-14  2019年10月26日 

      詳細を見る

      記述言語:英語   会議種別:口頭発表(招待・特別)  

      researchmap

    • どんどん見つかる例外的な遺伝暗号 招待有り

      向井崇人

      第41回⽇本分⼦⽣物学会年会  2018年11月28日 

      詳細を見る

      記述言語:日本語   会議種別:口頭発表(招待・特別)  

      researchmap

    • A new chassis tRNA for genetic code expansion? 国際会議

      Takahito Mukai, Dieter Söll

      AARS2017  2017年10月30日 

      詳細を見る

      記述言語:英語   会議種別:口頭発表(一般)  

      researchmap

    • A rare codon can be captured by a non-canonical amino acid. 国際会議

      Takahito Mukai, Kensaku Sakamoto

      The 25th tRNA Conference  2014年9月 

      詳細を見る

      記述言語:英語   会議種別:口頭発表(一般)  

      researchmap

    • 細胞の遺伝暗号改変と、これからの課題 招待有り

      向井崇人, 大竹和正, 坂本健作

      「細胞を創る」研究会5.0  2012年11月 

      詳細を見る

      記述言語:日本語   会議種別:口頭発表(一般)  

      researchmap

    ▼全件表示

    担当経験のある科目(授業)

    • 2020年5月 - 2020年7月 
      生命理学ゼミナール2 ( 立教大学 )

      詳細を見る

    •  
      生物化学2 ( 立教大学 )

      詳細を見る

    所属学協会

    • 2020年1月 - 現在 
      日本ゲノム微生物学会

      詳細を見る

    •  
      「細胞を創る」研究会

      詳細を見る

    共同研究・競争的資金等の研究

    • ゾンビ細胞を用いたゲノム入れ替え法の開発

      日本学術振興会  科学研究費助成事業 基盤研究(C) 

      向井 崇人

      詳細を見る

      2023年4月 - 2026年3月

      課題番号:23K05743

      配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

      researchmap

    • 立体構造タグを用いたtRNA分子群の操作法の開発

      科研費  研究活動スタート支援 

      向井崇人

      詳細を見る

      2018年8月 - 2020年3月

      担当区分:研究代表者  資金種別:競争的資金

      researchmap

    • セレノシステイン/システイン遺伝暗号の全様解明

      日本学術振興会  海外特別研究員 

      向井崇人

      詳細を見る

      2016年4月 - 2018年3月

      担当区分:研究代表者  資金種別:競争的資金

      researchmap

    • 非天然型アミノ酸を用いて酵素活性をデザインするための基幹技術開発

      理化学研究所  基礎科学特別研究員 

      向井崇人

      詳細を見る

      2012年4月 - 2014年5月

      担当区分:研究代表者  資金種別:競争的資金

      researchmap

    • セ ン ス ・ コ ド ン ・ リ ・ ア サ イ メ ン ト

      科研費  若手研究 (B) 

      向井崇人

      詳細を見る

      2012年4月 - 2014年3月

      担当区分:研究代表者  資金種別:競争的資金

      researchmap

    • 哺乳類細胞の遺伝暗号拡張によるタンパク質翻訳後修飾の細胞機能解明

      特別研究員奨励費(DC1) 

      向井崇人

      詳細を見る

      2008年4月 - 2011年3月

      担当区分:研究代表者  資金種別:競争的資金

      researchmap

    ▼全件表示

    産業財産権

    • 機能性DNAカセット及びプラスミド

      末次 正幸, 向井 崇人

      詳細を見る

      出願番号:特願2022-006523  出願日:2022年1月19日

      researchmap

    • COMPOSITIONS AND METHODS FOR MAKING SELENOCYSTEINE CONTAINING POLYPEPTIDES

      Dieter Söll, Caroline Aldag, Michael Hohn, Takahito Mukai

      詳細を見る

      出願番号:特願15/724678  出願日:2017年10月4日

      公開番号:特開20180105854  公開日:2018年4月19日

      特許番号/登録番号:特許US-10876142-B2  登録日:2020年12月29日 

      権利者:Yale University

      researchmap

    • COMPOSITIONS AND METHODS FOR MAKING SELENOCYSTEINE CONTAINING POLYPEPTIDES

      Dieter Söll, 向井崇人, HOFFMAN, Kyle

      詳細を見る

      出願番号:PCT/US2018/054437  出願日:2018年10月4日

      公開番号:特開WO/2019/071023  公開日:2019年4月11日

      researchmap

    • 非天然タンパク質製造用の組換え細菌の作製方法、及びその利用

      横山 茂之, 向井 崇人, 坂本 健作, 松元 明子

      詳細を見る

      出願人:国立研究開発法人理化学研究所

      出願番号:特願2012-520488  出願日:2011年6月16日

      特許番号/登録番号:特許第5858543号  発行日:2015年12月25日

      J-GLOBAL

      researchmap

    • アミノアシルtRNA合成酵素活性を有するポリペプチド及びその利用

      横山 茂之, 坂本 健作, 大木 健二, 向井 崇人

      詳細を見る

      出願人:独立行政法人理化学研究所

      出願番号:特願2009-024136  出願日:2009年2月4日

      公開番号:特開2009-207490  公開日:2009年9月17日

      特許番号/登録番号:特許第5585904号  発行日:2014年8月1日

      J-GLOBAL

      researchmap

    • エステル結合を含む非天然タンパク質の製造方法

      横山 茂之, 坂本 健作, 柳沢 達男, 向井 崇人, 小林 隆嗣

      詳細を見る

      出願人:独立行政法人理化学研究所

      出願番号:特願2009-542600  出願日:2008年11月21日

      特許番号/登録番号:特許第5419220号  発行日:2013年11月29日

      J-GLOBAL

      researchmap

    • サプレッサーtRNAの合成方法、DNA構築物及びそれを用いた非天然型アミノ酸組み込みタンパク質の製造

      横山 茂之, 坂本 健作, 樋野 展正, 向井 崇人, 小林 隆嗣

      詳細を見る

      出願人:独立行政法人理化学研究所

      出願番号:特願2008-502740  出願日:2007年2月22日

      特許番号/登録番号:特許第5196378号  発行日:2013年2月15日

      J-GLOBAL

      researchmap

    • METHOD FOR SYNTHESIS OF SUPPRESSOR tRNA, DNA CONSTRUCT, AND PRODUCTION OF PROTEIN HAVING NON-NATURAL AMINO ACID INTEGRATED THEREIN BY USING THE DNA CONSTRUCT

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

      詳細を見る

      特許番号/登録番号:特許EP1992698B1  発行日:2018年9月5日

      researchmap

    • Process for production of non-natural protein having ester bond therein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Tatsuo Yanagisawa, Takahito Mukai, Takatsugu Kobayashi

      詳細を見る

      特許番号/登録番号:特許US8785152B2  発行日:2014年7月22日

      researchmap

    • Process for production of non-natural protein having ester bond therein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Tatsuo Yanagisawa, Takahito Mukai, Takatsugu Kobayashi

      詳細を見る

      特許番号/登録番号:特許EP2221370B1  発行日:2014年4月16日

      researchmap

    • Polypeptide having activity of aminoacyl-tRNA synthetase and use thereof

      Shigeyuki Yokoyama, Kensaku Sakamoto, Kenji Oki, Takahito Mukai

      詳細を見る

      特許番号/登録番号:特許US8293512B2  発行日:2012年10月23日

      researchmap

    • Method for constructing recombinant bacterium for producing non-native protein, and utilization of same

      Shigeyuki Yokoyama, Takahito Mukai, Kensaku Sakamoto, Akiko Matsumoto

      詳細を見る

      特許番号/登録番号:特許US9340790B2  発行日:2016年5月17日

      researchmap

    • Method for constructing recombinant bacterium for producing non-native protein, and utilization of same

      Shigeyuki Yokoyama, Takahito Mukai, Kensaku Sakamoto, Akiko Matsumoto

      詳細を見る

      特許番号/登録番号:特許EP2584037B1  発行日:2016年8月10日

      researchmap

    • Method of synthesizing a suppressor tRNA, DNA construct and use thereof for producing a non-natural amino acid-incorporated protein

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

      詳細を見る

      特許番号/登録番号:特許US8822180B2  発行日:2014年9月2日

      researchmap

    • Method of synthesizing a suppressor TRNA, DNA construct and use thereof for producing a protein including a non-natural amino acid

      Shigeyuki Yokoyama, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi

      詳細を見る

      特許番号/登録番号:特許US8168407B2  発行日:2012年5月1日

      researchmap

    ▼全件表示

    メディア報道

    • 3分割したゲノムからなる大腸菌を作製、自由なゲノム出し入れを実現 ~モデル生物でのゲノムインストール技術~

      JST & 立教大学  2021年4月

      詳細を見る

    • Proving the Genetic Code’s Flexibility

      JGI  2016年3月

      詳細を見る

    • DNA情報の変換ルールを人為的に改変 -多様なアミノ酸を高い効率でタンパク質に導入することが可能に-

      理化学研究所  2015年5月

      詳細を見る

     

    お問い合わせ先
    外部の方
    学内の方