論文 - 川上　了史

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年05月

ISSN  10465928

　概要を見る

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.

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

Yamashita M., Kawakami N., Miyamoto K.

ChemPlusChem （ChemPlusChem）  88 （ 3 ）  2023年03月

　概要を見る

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.

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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年01月

ISSN  00027863

　概要を見る

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.

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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年10月

ISSN  13597345

　概要を見る

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.

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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年03月

　概要を見る

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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Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches

Furukawa M., Kawakami N., Tomizawa A., Miyamoto K.

Scientific Reports （Scientific Reports）  9 （ 1 ）  2019年12月

　概要を見る

Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that the low-crystallinity PET (lcPET) hydrolysis activity of thermostable cutinase from Thermobifida fusca (TfCut2), was increased by the addition of cationic surfactant that attracts enzymes near the lcPET film surface via electrostatic interactions. This approach was applicable to the mutant TfCut2 G62A/F209A, which was designed based on a sequence comparison with PETase from Ideonella sakaiensis. As a result, the degradation rate of the mutant in the presence of cationic surfactant increased to 31 ± 0.1 nmol min−1 cm−2, 12.7 times higher than that of wild-type TfCut2 in the absence of surfactant. The long-duration reaction showed that lcPET film (200 μm) was 97 ± 1.8% within 30 h, the fastest biodegradation rate of lcPET film thus far. We therefore believe that our approach would expand the possibility of enzyme utilization in industrial PET biodegradation.

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Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants

Furukawa M., Kawakami N., Oda K., Miyamoto K.

ChemSusChem （ChemSusChem）  11 （ 23 ） 4018 - 4025 2018年12月

ISSN  18645631

　概要を見る

Enzymatic degradation of poly(ethylene terephthalate) (PET) is promising because this process is safer than conventional industrial approaches. Recently, a cationic PET hydrolase (PETase) was identified from Ideonella sakaiensis. Pre-incubation of a low-crystallinity PET film with anionic surfactants prior to initiating the reaction was found to improve PETase activity 120-fold. After 36 h at 30 °C, the film thickness decreased by 22 %. The binding of surfactants to the film makes the surface anionic, thereby attracting the cationic PETase. Mutagenesis of PETase showed that the surface cationic region formed by R53, R90, and K95, which are located on the same side as the substrate binding pocket, was crucial for efficient acceleration of activity by the anionic surfactant. Thus, surfactant bound on PET aligns the orientation of the active site to the surface, resulting in efficient hydrolysis. We believe that this approach using PETase could be further improved by designing surfactant molecules for the more efficient enzymatic PET degradation.

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Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces

川上 了史

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  57 （ 38 ） 12400 - 12404 2018年09月

研究論文（学術雑誌）,  ISSN  1433-7851

　概要を見る

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

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Protein Nanoparticle Formation Using a Circularly Permuted alpha-Helix Rich Trimeric Protein

川上 了史

BIOCONJUGATE CHEMISTRY 28 （ 2 ） 336 - 340 2017年02月

研究論文（学術雑誌）,  ISSN  1043-1802

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Activation of Wild-Type Cytochrorne P450BM3 by the Next Generation of Decoy Molecules: Enhanced Hydroxylation of Gaseous Alkanes and Crystallographic Evidence

川上 了史

ACS CATALYSIS 5 （ 1 ） 150 - 156 2015年01月

研究論文（学術雑誌）,  ISSN  2155-5435

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Modulating the catalytic activity and the substrate specificity of alcohol dehydrogenases using cyclic ethers

川上 了史

CATALYSIS SCIENCE & TECHNOLOGY 5 （ 8 ） 3922 - 3925 2015年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2044-4753

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Draft genome sequence of Bordetella bronchiseptica KU1201, the first isolation source of arylmalonate decarboxylase

川上 了史

Genome Announcements 2015年

研究論文（学術雑誌）, 共著, 査読有り

Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules

川上 了史

CHEMICAL SCIENCE 4 （ 6 ） 2344 - 2348 2013年

研究論文（学術雑誌）,  ISSN  2041-6520

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Highly Selective Hydroxylation of Benzene to Phenol by Wild-type Cytochrome P450BM3 Assisted by Decoy Molecules

川上 了史

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 52 （ 26 ） 6606 - 6610 2013年

研究論文（学術雑誌）,  ISSN  1433-7851

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Chiral-Substrate-Assisted Stereoselective Epoxidation Catalyzed by H2O2-Dependent Cytochrome P450SPa

川上 了史

CHEMISTRY-AN ASIAN JOURNAL 7 （ 10 ） 2286 - 2293 2012年10月

研究論文（学術雑誌）,  ISSN  1861-4728

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Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells

川上 了史

CHEMBIOCHEM 13 （ 14 ） 2045 - 2047 2012年09月

研究論文（学術雑誌）,  ISSN  1439-4227

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Use of Perfluorocarboxylic Acids To Trick Cytochrome P450BM3 into Initiating the Hydroxylation of Gaseous Alkanes

川上 了史

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 50 （ 23 ） 5315 - 5318 2011年

研究論文（学術雑誌）,  ISSN  1433-7851

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Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

川上 了史

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1790 （ 10 ） 1327 - 1333 2009年10月

研究論文（学術雑誌）,  ISSN  0304-4165

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A novel vanadium reductase, Vanabin2, forms a possible cascade involved in electron transfer

川上 了史

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1794 （ 4 ） 674 - 679 2009年04月

研究論文（学術雑誌）,  ISSN  1570-9639

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Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea

川上 了史

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1760 （ 7 ） 1096 - 1101 2006年07月

研究論文（学術雑誌）,  ISSN  0304-4165

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