Kawakami, Norifumi

写真a

Affiliation

Faculty of Science and Technology, Department of Biosciences and Informatics (Yagami)

Position

Assistant Professor/Senior Assistant Professor

External Links

Career 【 Display / hide

  • 2009.04
    -
    2013.03

    名古屋大学, 物質科学国際研究センター

  • 2013.04
    -
    2014.03

    名古屋大学, 大学院理学研究科生命理学専攻

  • 2014.04
    -
    2017.03

    慶應義塾大学, 理工学部

  • 2017.04
    -
    2020.03

    慶應義塾大学, 理工学部, 専任講師(有期)

  • 2020.04
    -
    Present

    慶應義塾大学, 理工学部

Academic Background 【 Display / hide

  • 2002.04
    -
    2004.03

    Ube National College of Technology, 専攻科物質科学専攻

    Technical College, Completed

  • 2004.04
    -
    2006.03

    Hiroshima University, 大学院理学研究科博士課程前期

    Master's course

  • 2006.04
    -
    2009.03

    Hiroshima University, 大学院理学研究科博士課程後期

    Doctoral course

Academic Degrees 【 Display / hide

  • 博士(理学), Hiroshima University, Coursework, 2009.03

Licenses and Qualifications 【 Display / hide

  • 放射線取扱主任者第一種, 2015.03

 

Research Areas 【 Display / hide

  • Natural Science / Biogeosciences (金属イオン、金属タンパク質、実験室進化)

  • Nanotechnology/Materials / Chemistry and chemical methodology of biomolecules (タンパク質、ナノ粒子、ハイドロゲル)

Research Themes 【 Display / hide

  • 生命の元素に対する進化的可塑性の解明, 

    2015.04
    -
    Present

  • タンパク質を鋳型とした分子構造の設計と材料への応用, 

    2014.03
    -
    Present

 

Papers 【 Display / hide

  • Column-free purification of an artificial protein nanocage, TIP60

    Nasu E., Kawakami N., Ohara N., Hayashi K., Miyamoto K.

    Protein Expression and Purification (Protein Expression and Purification)  205 2023.05

    ISSN  10465928

     View Summary

    Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification. TIP60, which has an anionic surface charge, aggregated at mildly acidic pH and redissolved at neutral pH, maintaining its cage structure. This pH-responsive reversible precipitation allowed us to purify TIP60 from soluble fractions of the E. coli cell lysate by controlling the pH. Compared with conventional Ni-NTA column purification, the pH-responsive precipitation method provided purified TIP60 with similar purity (∼80%) and higher yield. This precipitation purification method should facilitate the large-scale investigation and practical use of TIP60 nanocages.

  • Hydrophobization of a TIP60 Protein Nanocage for the Encapsulation of Hydrophobic Compounds

    Yamashita M., Kawakami N., Miyamoto K.

    ChemPlusChem (ChemPlusChem)  88 ( 3 )  2023.03

     View Summary

    Encapsulation of hydrophobic molecules in protein-based nanocages is a promising approach for dispersing these molecules in water. Here, we report a chemical modification approach to produce a protein nanocage with a hydrophobic interior surface based on our previously developed nanocage, TIP60. The large pores of TIP60 act as tunnels for small molecules, allowing modification of the interior surface by hydrophobic compounds without nanocage disassembly. We used four different hydrophobic compounds for modification. The largest modification group tested, pyrene, resulted in a modified TIP60 that could encapsulate aromatic photosensitizer zinc phthalocyanine (ZnPC) more efficiently than the other modification compounds. The encapsulated ZnPC generated singlet oxygen upon light activation in the aqueous phase, whereas ZnPC alone formed inert aggregates under the same experimental conditions. Given that chemical modification allows a wider diversity of modifications than mutagenesis, this approach could be used to develop more suitable nanocages for encapsulating hydrophobic molecules of interest.

  • Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions

    Ohara N., Kawakami N., Arai R., Adachi N., Moriya T., Kawasaki M., Miyamoto K.

    Journal of the American Chemical Society (Journal of the American Chemical Society)  145 ( 1 ) 216 - 223 2023.01

    ISSN  00027863

     View Summary

    Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

  • Icosahedral 60-meric porous structure of designed supramolecular protein nanoparticle TIP60

    Obata J., Kawakami N., Tsutsumi A., Nasu E., Miyamoto K., Kikkawa M., Arai R.

    Chemical Communications (Chemical Communications)  57 ( 79 ) 10226 - 10229 2021.10

    ISSN  13597345

     View Summary

    Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self-assembled from fusion proteins of a pentameric Sm-like protein and a dimeric MyoX-coil domain. Herein, we report the icosahedral 60-meric structure of TIP60 solved using single-particle cryo-electron microscopy. Interestingly, the structure revealed 20 regular-triangle-like pores on the surface. TIP60 and its mutants have many modifiable sites on their exterior and interior surfaces. The TIP60 architecture will be useful in the development of biomedical and biochemical nanoparticles/nanocages for future applications.

  • Nanopore-Controlled Dual-Surface Modifications on Artificial Protein Nanocages as Nanocarriers

    Nasu E., Kawakami N., Miyamoto K.

    ACS Applied Nano Materials (ACS Applied Nano Materials)  4 ( 3 ) 2434 - 2439 2021.03

     View Summary

    Chemical modification of the interior and exterior surfaces of protein nanocages holds promise for various applications such as cosmetics, pharmaceuticals, and catalysts. However, dual-surface modification of these surfaces using different chemicals remains challenging, particularly when the same substituents, such as cysteine thiols, are modified. We recently produced an artificial protein nanocage called TIP60 that has 20 large surface pores. Chemical modification of cysteine residues introduced in the interior surface of TIP60 showed that these pores allow the passage of small molecules from the outside environment to the inside of the nanocage. In this study, we found that the surface pores on TIP60 function as size-dependent molecular filters. Modification experiments using different-sized polymers containing maleimide groups, which specifically react with thiols, showed that macromolecules with diameters larger than that of the pores could not penetrate into the inner cavity. This molecular size discrimination by the pores prompted us to perform stepwise dual-surface functionalization of a double mutant of TIP60 presenting cysteine residues on the interior and exterior surfaces. This was achieved by modifying the exterior cysteine residues with a polymer containing a maleimide group that cannot penetrate to the inside of the nanocage, followed by modification of the interior cysteine residues using thiol-containing small molecules. Dual-functionalized TIP60 released internal small molecules in a redox-responsive manner. This simple approach for dual-surface modification would make TIP60 a useful nanocarrier for a broad range of applications including drug-delivery and molecular filtration systems.

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Papers, etc., Registered in KOARA 【 Display / hide

Research Projects of Competitive Funds, etc. 【 Display / hide

  • 実験室進化で生じた大腸菌変異株の鉄輸送体に着目した進化プロセスの検証

    2022.04
    -
    2024.03

    Research grant, Principal investigator

  • 大腸菌実験室進化を通じた生物の元素利用に対する進化的可塑性の検討

    2021.07
    -
    2024.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), Principal investigator

  • 人工球状タンパク質超分子を用いたナノスケール相分離の実現とその応用

    2018.04
    -
    2021.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Principal investigator

Awards 【 Display / hide

  • 第8回新化学技術研究奨励賞 新化学技術推進協会

    2019.06

    Type of Award: Award from publisher, newspaper, foundation, etc.

  • 日本蛋白質科学会若手奨励賞優秀賞

    2017.06

 

Courses Taught 【 Display / hide

  • TOPICS IN BIOSCIENCES AND INFORMATICS 2

    2023

  • THE LIBERAL ARTS OF LIFE

    2023

  • SEMINAR IN BIOSCIENCES AND INFORMATICS

    2023

  • LABORATORY IN SCIENCE

    2023

  • INTRODUCTION TO BIOLOGY TODAY

    2023

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Courses Previously Taught 【 Display / hide

  • 生体反応論第2

    Keio University

    2017.04
    -
    Present

    Spring Semester