Details of a Researcher - Fujiwara, Kei

Papers - Fujiwara, Kei

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Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Aye Seaim Lwin, Fujiwara Kei, Ueki Asuka, Doi Nobuhide

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 499 ( 2 ) 170 - 176 2018.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

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Increasing Elasticity through Changes in the Secondary Structure of Gelatin by Gelation in a Microsized Lipid Space

Sakai Atsushi, Murayama Yoshihiro, Fujiwara Kei, Fujisawa Takahiro, Sasaki Saori, Kidoaki Satoru, Yanagisawa Miho

ACS Central Science (ACS Central Science) 4 ( 4 ) 477 - 483 2018.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2374-7943

Even though microgels are used in a wide variety of applications, determining their mechanical properties has been elusive because of the difficulties in analysis. In this study, we investigated the surface elasticity of a spherical microgel of gelatin prepared inside a lipid droplet by using micropipet aspiration. We found that gelation inside a microdroplet covered with lipid membranes increased Young's modulus E toward a plateau value E∗ along with a decrease in gel size. In the case of 5.0 wt % gelatin gelled inside a microsized lipid space, the E∗ for small microgels with R ≤ 50 μm was 10-fold higher (35-39 kPa) than that for the bulk gel (∼3 kPa). Structural analysis using circular dichroism spectroscopy and a fluorescence indicator for ordered beta sheets demonstrated that the smaller microgels contained more beta sheets in the structure than the bulk gel. Our finding indicates that the confinement size of gelling polymers becomes a factor in the variation of elasticity of protein-based microgels via secondary structure changes.

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Single Micrometer-Sized Gels: Unique Mechanics and Characters for Applications.

Yanagisawa Miho, Watanabe Chiho, Fujiwara Kei

Gels (MDPI) 4 ( 2 ) 29 2018.03

Research paper (scientific journal), Joint Work, Accepted

Microgels—small gels of submicron to micron size—are widely used in food, cosmetics and biomedical applications because of their biocompatibility and/or fast response to external environments. However, the properties of “single” microgels have not been characterized due to limitations in preparation technologies and measurement methods for single microgels with sizes in the multi-micrometer range. The synthesis of multiple shapes of single microgels and their characterization are important for further functionalization and application of gel-based materials. In this review, we explain the recent advancements in microgel fabrication and characterization methods for single microgels. The first topic discussed includes the self-assembly methods for single microgel fabrication using physical phenomena such as phase separation, interfacial wetting and buckling instability. The second topic deals with methods for analyzing the mechanics of single microgels and the differences between their mechanical characteristics and those of bulk gels. The recent progress in the fabrication and characterization of single microgels will bring important insights to the design and functionalization of gel-based materials.

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Artificial Cell Fermentation as a Platform for Highly Efficient Cascade Conversion

Fujiwara Kei, Adachi Takuma, Doi Nobuhide

ACS Synthetic Biology (ACS Synthetic Biology) 7 ( 2 ) 363 - 370 2018.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 2161-5063

Because of its high specificity and stereoselectivity, cascade reactions using enzymes have been attracting attention as a platform for chemical synthesis. However, the sensitivity of enzymes outside their optimum conditions and their rapid decrease of activity upon dilution are drawbacks of the system. In this study, we developed a system for cascade enzymatic conversion in bacteria-shaped liposomes formed by hypertonic treatment, and demonstrated that the system can overcome the drawbacks of the enzymatic cascade reactions in bulk. This system produced final products at a level equivalent to the maximum concentration of the bulk system (0.10 M, e.g., 4.6 g/L), and worked even under conditions where enzymes normally lose their function. Under diluted conditions, the conversion rate of the artificial cell system was remarkably higher than that in the bulk system. Our results indicate that artificial cells can behave as a platform to perform fermentative production like microorganisms.

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Improvement of microtas for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Einaga Y., Matsumoto Y., Miki N., Fujiwara K., Doi N.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 (22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018) 4 2247 - 2249 2018

Here we report an improved μTAS based on electrochemical measurements and microbeads sorting for screening of NAD(P)-dependent oxidoreductases. By using this system, we measured the activity of model enzymes immobilized on microbeads by detection of NADH with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. Then, we recovered the magnetic microbeads or the droplets containing genes of enzymes with higher activity by magnetophoretic or dielectrophoretic sorting, respectively. Our μTAS should be useful for directed evolution of various NAD(P)-dependent oxidoreductases with high activity and stability in the future.

Universal glass-forming behavior of in vitro and living cytoplasm

Nishizawa Kenji, #HFujiwara Kei#IR, Ikenaga Masahiro, Nakajo Nobushige, Yanagisawa Miho, Mizuno Daisuke

Scientific Reports 7 ( 1 ) 15143 2017.12

Research paper (scientific journal), Joint Work, ISSN 2045-2322

Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of in vitro and living cytoplasm using the particle-tracking and manipulation technique. The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart.

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Liposomal internal viscosity affects the fate of membrane deformation induced by hypertonic treatment

Fujiwara Kei, Yanagisawa Miho

Soft Matter (Royal Society of Chemistry) 13 ( 48 ) 9192 - 9198 2017.11

Research paper (scientific journal), Joint Work, ISSN 1744-683X

Artificial lipid membranes have been utilized to understand the physical mechanisms of the deformation patterns of live cells. However, typical artificial membrane systems contain only dilute components compared to those in the cytoplasm of live cells. By using giant unilamellar liposomes containing dense protein solutions similar to those in live cells, we here reveal that viscosity derived from internal crowding affects the deformation patterns of lipid membranes. After hypertonic treatment, liposome deformation patterns transitioned from budding to tubing when the initial internal macromolecular concentrations were increased. Remarkably, instead of observing different transition concentrations between two species of macromolecules, the viscosity at the transition concentration was found to be similar. Further analyses clearly demonstrated that the internal viscosity affects the deformation patterns of lipid membranes induced by hypertonic treatment. These results indicate that the viscosity of the cytoplasm is a key factor in determining cell deformation, and suggest the association of a process involving dynamic instability, such as a viscous fingering phenomenon, during the determination of deformation patterns by hypertonic treatment.

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In vitro transcription-translation using bacterial genome as a template to reconstitute intracellular profile

Fujiwara Kei, Sawamura Tsunehito, Niwa Tatsuya, Deyama Tatsuki, Nomura Shin Ichiro M., Taguchi Hideki, Doi Nobuhide

Nucleic Acids Research 45 ( 19 ) 11449 - 11458 2017.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

In vitro transcription-translation systems (TX-TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX-TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription-translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC-MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX-TL using genome as a template.

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DNA cytoskeleton for stabilizing artificial cells

Kurokawa Chikako, Fujiwara Kei, Morita Masamune, Kawamata Ibuki, Kawagishi Yui, Sakai Atsushi, Murayama Yoshihiro, Nomura M. Shin-ichiro, Murata Satoshi, Takinoue Masahiro, and Yanagisawa　Miho

Proceedings of the National Academy of Sciences (National Academy of Sciences) 114 ( 28 ) 7228 - 7233 2017.06

Research paper (scientific journal), Joint Work, Accepted

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

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Human-derived fusogenic peptides for the intracellular delivery of proteins

Sudo Kei, Niikura Keisuke, Iwaki Kouta, Kohyama Shunshi, Fujiwara Kei, Doi Nobuhide

Journal of Controlled Release 255 1 - 11 2017.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0168-3659

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322–340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and β-galactosidase were delivered efficiently and retained their activities.

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In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display

Nakayama Masanao, Komiya Shoko, FUJIWARA Kei, Horisawa kenichi, Doi Nobuhide

Biochem. Biophys. Res. Comm. 478 ( 2 ) 606 - 611 2016.09

Research paper (scientific journal), Joint Work, Accepted

Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption

FUJIWARA Kei, Doi Nobuhide

PLOS ONE 11 ( 4 ) e0154614 2016.04

Research paper (scientific journal), Joint Work, Accepted

Identification of novel in vivo obligate GroEL/ES substrates based on data from a cell-free proteomics approach.

Niwa Tatsuya, FUJIWARA Kei, Taguchi hideki

FEBS Lett. 590 ( 2 ) 251 - 257 2016.01

Research paper (scientific journal), Joint Work, Accepted

PURE mRNA display for in vitro selection of single-chain antibodies.

Nagumo Yu, FUJIWARA Kei, Horisawa Kenichi, Yanagawa Hiroshi, Doi Nobuhide

J. Biochem. 159 ( 5 ) 519 - 526 2015.12

Research paper (scientific journal), Joint Work, Accepted

Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions

Morita Masamune, Onoe Hiroaki, Yanagisawa Miho, Ito Hiroaki, Ichikawa Masatoshi, Fujiwara Kei, Saito Hirohide, Takinoue Masahiro

ChemBioChem 16 ( 14 ) 2029 - 2035 2015.09

Research paper (scientific journal), Joint Work, ISSN 1439-4227

We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2, μL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems. Shoot to fill: We have developed a simple and robust microfluidic method for the generation of cell-sized liposomes with controlled lipid composition. Microdroplets are generated by centrifugal microdroplet shooting then filtering to form liposomes. We believe that this method can be applied to the study of cell-sized liposomes and the construction of artificial cell-like systems.

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Generation of monodisperse cell-sized microdroplets using a centrifuge-based axisymmetric co-flowing microfluidic device

Hitoyoshi Yamashita, Masamune Morita, Haruka Sugiura, Kei Fujiwara, Hiroaki Onoe, Masahiro Takinoue

Journal of Bioscience and Bioengineering (Elsevier) 119 ( 4 ) 492 - 495 2015.04

Research paper (scientific journal), Joint Work, Accepted

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Peptide sequences converting polyglutamine into a prion in yeast

Wataru Odani, Kazuhiro Urata, Momoko Okuda, Shunsuke Okuma, Hiroko Koyama, Chan‐Gi Pack, Kei Fujiwara, Tatsuya Nojima, Masataka Kinjo, Shigeko Kawai‐Noma, Hideki Taguchi

FEBS Journal 282 ( 3 ) 477 - 490 2015.02

Research paper (scientific journal), Joint Work, Accepted

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Development of μtas for high-throughput screening of nad(p)-dependent oxidoreductases

Shimokihara Shota, Shitara Shunya, Oyobiki Ryo, Watanabe Takeshi, Einaga Yasuaki, Matsumoto Yoshinori, Fujiwara Kei, Horisawa Kenichi, Doi Nobuhide

1302 - 1304 2015

Research paper (international conference proceedings), Joint Work

Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activity is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we developed a microfluidic total analysis system for high-throughput screening of NAD(P)-dependent oxidoreductases. This system comprises three microfluidic devices: an encapsulating device, a NADH measuring device, and a magnetic beads sorting device. We demonstrated the utility of each part of the system.

Conversion of a Chaperonin GroEL-Independent Protein into an Obligate Substrate

ISHIMOTO takuya, FUJIWARA Kei, NIWA Tatsuya, TAGUCHI hideki

The Journal of Biological Chemistry (The American Society for Biochemistry and Molecular Biology, Inc.) 289 32073 - 32080 2014.11

Research paper (scientific journal), Joint Work

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Multiple patterns of polymer gels in microspheres due to the interplay among phase separation, wetting, and gelation

Miho Yanagisawa, Shinpei Nigorikawa, Takahiro Sakaue, Kei Fujiwara, and Masayuki Tokita

Proceedings of the National Academy of Sciences (National Acad Sciences) 111 ( 45 ) 15894 - 15899 2014.11

Research paper (scientific journal), Joint Work, Accepted

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