Satoshi Yamaguchi

The University of Tokyo, Edo, Tōkyō, Japan

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Publications (62)127.24 Total impact

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    ABSTRACT: Cell patterning on photo-responsive materials are a promising tool for preparing unique single-cell arrays. However, most conventional single-cell arrays on such smart materials can be applied only to adherent cells and limit cellular functions such as extension and migration within the patterned adhesive surfaces. In this study, a versatile single cell array that works with both non-adherent and adherent cells was constructed using a photo-cleavable polyethylene glycol (PEG)-lipid/collagen surface. On this single-cell array, cells behaved similar to their native functions without limitation from the patterned surface. Furthermore, quantitative imaging analyses of cellular motility and morphological changes were performed in a high-throughput manner.
    Macromolecular Bioscience 09/2014; · 3.65 Impact Factor
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    ABSTRACT: G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one-tenth in response to the extracellular acidic pH, and this acidic pH-induced G2A accumulation on cell surfaces may be explained by proton-induced dissociation of G2A from endocytic machinery.- Lan, W., Yamaguchi, S., Yamamoto, T., Yamahira, S., Tan, M., Murakami, N., Zhang, J., Nakamura, M., Nagamune, T. Visualization of the pH-dependent dynamic distribution of G2A in living cells.
    The FASEB Journal 06/2014; · 5.48 Impact Factor
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    ABSTRACT: Cell micropatterning methods with stimuli-responsive dynamic surfaces are getting a lot of attention in a wide variety of research fields, ranging from cell engineering to fundamental studies in cell biology. The surface of a slide coated with photo-cleavable poly(ethylene glycol) (PEG)-lipid can be used to spatiotemporally control cell immobilization and release by light irradiation. On the basis of this surface, it is easy to design simple methods for making a fine micropattern of any kind of cell. Furthermore, target cells can be selectively and rapidly released from this surface by light irradiation. In this review, we first describe how to obtain the photo-cleavable PEG-lipid from commercially available compounds through a facile four-step synthesis. Next, as a cell-patterning method, the protocols of coating substrates with the PEG-lipid, irradiating a pattern of light onto the coated substrate, and loading cells onto the irradiated surface are described. These protocols require no expensive equipment and potentially apply to any substrates that can adsorb serum albumin or chemically expose amine moieties on their surfaces. Finally, as an advanced method, cell release from the PEG-lipid surface in microfluidic devices is introduced. We also discuss the advantages and the possible applications of the present dynamic cell-patterning method.
    Methods in cell biology 01/2014; 120C:131-144. · 1.44 Impact Factor
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    ABSTRACT: Small enough to permeate through tumor blood vessel, and can be detect by ultrasound, phase change nano droplet (PCND) have been studied as contrast agents and therapeutic sensitizer for career. To investigate performance for these purpose, we investigated physical behavior of PCND, especially a lifetime of microbubbles generated by ultrasound stimulation. To investigate bubble's behavior after phase change, we observed a time-lapse change of bubbles population with following method. Focused transducer with a frequency of 3.3 MHz was placed in a water bath of 37 degrees. At the focal point, polyacrylamide gel including PCND was placed. Focused hydrophone was placed perpendicularly to the direction of ultrasound propagation. Two kind of ultrasound pulse wave was used; phase change pulse at the beginning and observation pulse at every 500 μs. The amplitude of scattering signal (SS) reflects the sum of scattering cross-section of bubbles. The time lapse observation of PCND after phase change showed two kinds of behavior, quick and slow decay of scattering signal from the bubbles. The unique increase of SS from the phase-changed bubbles in the monitoring phase was observed. We improved the experimental setup to measure bubble's population properly from various directions using 1-D array transducer and will present the result.
    The Journal of the Acoustical Society of America 11/2013; 134(5):4050. · 1.65 Impact Factor
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    ABSTRACT: We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (PEG)-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an inkjet printer, and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were incubated at 37°C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 07/2013; · 4.16 Impact Factor
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    ABSTRACT: We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG2-3mD conjugate, but was dramatically reduced in the biotin-PEG11-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG11, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG2-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.
    Journal of Bioscience and Bioengineering 05/2013; · 1.74 Impact Factor
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    ABSTRACT: Cell surface display of functional proteins is a powerful and useful tool for regulating and reinforcing cellular functions. Direct incorporation of site-specifically lipidated proteins from the extracellular medium is more rapid, easily controllable and reliable in displaying active proteins than expression through gene transfer. However, undesirable amphiphilic reagents such as organic cosolvents and detergents were required for suppressing aggregation of ordinary lipidated proteins in solution. We report here sortase A-catalyzed modification of proteins with a poly(ethylene glycol)(PEG)-lipid in situ on the surface of living cells. Proteins fused with a recognition tag were site-specifically ligated with the PEG-lipid which was preliminary incorporated into cell membranes. Accordingly, target proteins were successfully displayed on living cells without aggregation under an amphiphilic reagent-free condition. Furthermore, to demonstrate the availability of the present method, Fc domains of immunoglobulin G were displayed on cancer cells, and the phagocytosis of cancer cells with dendritic cells were enhanced through the Fc-Fc receptor interaction. Thus, the present facile chemoenzymatic method for protein display can be utilized for modulating cell-cell interactions in cell and tissue engineering fields. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 04/2013; · 4.16 Impact Factor
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    ABSTRACT: Streptococcus pyogenes sortase A (SpSrtA) was transduced into mammalian cells. Then SpSrtA-mediated intracellular circularization of a model protein was confirmed in a time and dose-dependent manner by Western blotting analysis. Direct transduction of SpSrtA is expected to be a strong tool for more conditional specific protein modification in mammalian cells.
    Journal of Bioscience and Bioengineering 04/2013; · 1.74 Impact Factor
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    ABSTRACT: A method for caging a target protein with steric regulator molecules was developed to enable simple, strict, light-induced control of protein activity.
    Chemical Communications 03/2013; · 6.38 Impact Factor
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    ABSTRACT: We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.
    Journal of Bioscience and Bioengineering 01/2013; · 1.74 Impact Factor
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    ABSTRACT: Analysis of the structural features of rhodopsin-type G-protein-coupled receptors (GPCRs) revealed the existence of an additional α-helix, termed helix 8, in the C-terminal tail. Furthermore, these GPCRs were determined to possess several conserved residues in their transmembrane domains. The functional deficiencies of receptors in which these domains or residues have been mutated have not been examined in living cells due to their accumulation in the endoplasmic reticulum (ER), although the ligand affinities of these receptors have been tested in membrane preparations. Recent studies have demonstrated that ER-accumulated receptors are effectively exported from ER using membrane permeable ligands as pharmacological chaperones. Here, we identified several residues of the platelet-activating factor receptor and leukotriene B(4) type-II receptor that are crucial for export from ER. Moreover, we used their specific ligands as pharmacological chaperones to traffic ER-accumulated GPCRs to the cell surface in order to examine the functional deficiencies of each mutant receptor. Here, we introduce the novel technique of site-specific N-terminal labeling of cell surface proteins in living cells with Sortase-A, a transpeptidase isolated from Staphylococcus aureus, to evaluate the trafficking of receptors after agonist stimulation.
    Methods in enzymology 01/2013; 521:203-16. · 1.90 Impact Factor
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    ABSTRACT: In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-b.
    Biotechnology Journal 09/2012; · 3.71 Impact Factor
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    ABSTRACT: A simple method for attaching immunoglobulin G (IgG) on the cell surface was successfully developed for enhancing phagocytosis of apoptotic tumor cells (ATCs) by dendritic cells (DCs) ex vivo. By conjugating with a poly(ethylene glycol) (PEG)-lipid, named the biocompatible anchor for the membrane (BAM), arbitrary IgG could be incorporated into the cell membrane. In particular, when IgG-BAM conjugates were prepared at the optimal molar ratio of IgG to BAM (1 to 20), almost all cells were efficiently modified with IgG by treatment with IgG-BAM. This simple method was successfully applied to four types of mammalian cells. Furthermore, treatment of ATCs with the IgG-BAM conjugate increased the phagocytosis ratio of ATCs by DCs two-fold when compared to no treatment. This phagocytosis-enhancing effect was nearly identical to treatment with a tumor-specific IgG. Thus, without employing the tumor-specific IgG, which is difficult to obtain for any tumor cells and is expensive, the present method could opsonize ATC with the use of arbitrary IgG. The results strongly indicate that IgG-BAM treatment represents a promising method for opsonizing ATC with human serum IgG, and that this approach will lead to objective clinical responses in DC vaccines.
    Pharmaceuticals 01/2012; 5(5):405-416.
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    ABSTRACT: Cells in the spotlight: A substrate was coated with a poly(ethylene glycol) (PEG) segment bound to a lipid through a linker that was cleaved by UV irradiation to leave a PEG-coated area. Irradiation of an arbitrary pattern easily and rapidly yielded a high-contrast cell pattern. Only the targeted cells on the cell pattern were selectively detached without cell damage on a microfluidic device.
    Angewandte Chemie International Edition 11/2011; 51(1):128-31. · 11.34 Impact Factor
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    ABSTRACT: Regulation of enzyme activity by biding a ligand or protein, such as allosteric enzyme and signal transduction, is common in vivo. This enzyme function is attractive from a view of application for bio reactor, DDS, and bio sensor. Some research groups developed conjugation of enzyme and stimuli responsive polymer to control enzyme activity or specific binding by stimulus such as temperature, pH, and light for affinity separation, microfluidic protein analysis and capture[1-3]. However, in order to control the enzyme activity by molecular recognition, the problem is that how to modify the surface of the enzyme to create the binding site for another ligand and how to control the activity of the enzyme when the ligand is recognized. There are some previous studies controlling enzyme activity by genetic modification[4] or antigen-antibody complex reaction[5]. These approaches utilize conformational change of enzyme with genetic modification or protein-ligand interaction. However, the reaction mechanism is complicated and is mostly difficult to know, thus we must repeat the genetic modification process and change the position of binding site by trial and error approach. The approach for making molecular recognition enzyme in previous researches also lacks versatility. Our approach to develop novel functional enzyme is that molecular recognition moiety is conjugated only near active pocket of the mutant enzyme. In this research, Cytochrome P450cam was used as enzyme and biotin with spacer arm was used as molecular recognition moiety. This enzyme regulates its activity by utilizing avidin-biotin affinity interaction as the following. In the absence of avidin, there is no steric hindrance and the enzymatic reaction proceeds normally. On the other hand, in the presence of avidin, the avidin that binds to biotin covers the active pocket of the enzyme, then enzyme reaction does not proceed by the inhibition of substrate diffusion. Therefore, the enzyme can control activity itself in response to specific molecule. The advantage of this approach is that we can design the required enzymatic function by changing both enzyme and molecular recognition moiety. In this study, we firstly fabricated genetically modified P450cam that has one thiol group near its active pocket, abbreviated as 3mD. This mutant has a property that thiol-reacticve pegylated biotin is able to be conjugated only at targeted position. Then, conjugation of 3mD and pegylated biotin with different length of spacer arm was performed. Finally, the concept of this study was proven and the effect of the length of spacer arm on the avidin signal response was also examined. The activity of 3mD was retained compared to that of wild type P450cam. Furthermore, 3mD kept their tertial structure after genetic modification by measuring absorption peak of ferrous CO-complexed state[6]. This shows that 3mD maintained enough activity without any denaturation. The number of thiol group on the surface of 3mD were investigated. From thiol group determination, about 0.7 mol of thiol groups existed in 1 mol of 3mD, while thiol group on the surface of wild type P450cam was not detected. This result is close to theoretical value of 3mD. These results suggest that 3mD was prepared intendedly. Then, three kinds of pegylated biotins that have different length of spacer arm were conjugated to 3mD. The length of spacer arms are 39 atoms (biotin-PEG11), 12 atoms (biotin-PEG2), and 2 atoms (biotin-Alkyl2) respectively. This conjugation was confirmed by determining the amount of thiol groups before and after biotinylation and about 70% of thiol groups were successfully biotinylated. Finally, we evaluated each activity of biotinylated 3mD before and after avidin binding. In the case of biotin-PEG11-3mD, which has the longest spacer arm, the activity did not change when avidin binds to biotin. On the other hand, the activity of 3mD with shorter spacer arm, especially biotin-Alkyl2-3mD, dramatically decreased to 60% after avidin binding. The diameter of the active pocket is around 17 angstrom, which is almost the same as the length of spacer arm in biotin-Alkyl2-3mD. The conjugation site exists on the circumference of this pocket. As for biotin-PEG11-3mD, bound avidin was too far from the active pocket of 3mD to cover the pocket. Meanwhile, as the length of spacer arm is shorter, bound avidin get closer to the active pocket, and the avidin appropriately covered the active pocket in biotin-Alkyl2-3mD. We can conclude that we successfully developed novel functional enzyme and optimized its nanostructure. References [1] A. S. Hoffman, Clinical Chemistry 46, 1478 (2000). [2] A. S. Hoffman et al., Journal of Biomedical Materials Research 52, 577 (2000). [3] C. D. H. Alarcon, S. Pennadam, C. Alexander, Chemical Society Reviews 34, 276 (2005). [4] F. T. K. Lau, A. R. Fersht, Nature 326, 811 (1987). [5] C. A. Brennan, K. Christianson, M. A. Lafleur, W. Mandecki, Proceedings of the National Academy of Sciences of the United States of America 92, 5783 (1995). [6] S. A. Martinis et al., Biochemistry 35, 14530 (1996).
    2011 AIChE Annual Meeting; 10/2011
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    ABSTRACT: Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.
    Biotechnology and Bioengineering 08/2011; 109(1):244-51. · 4.16 Impact Factor
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    ABSTRACT: A Staphylococcus aureus transpeptidase, sortase A (SrtA), catalyzes selective peptide/protein ligations that have been applied to cell imaging and protein engineering, while the ligations do not proceed to completion due to their reversibility. We successfully enhanced SrtA-mediated protein ligation through the formation of a β-hairpin around the ligation site.
    Chemical Communications 03/2011; 47(16):4742-4. · 6.38 Impact Factor
  • Etsushi Yamamoto, Satoshi Yamaguchi, Teruyuki Nagamune
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    ABSTRACT: An important property of ionic liquids consisting of cations and anions is that the chemical structures can be easily tuned. To expand the repertoire of effective ionic liquid-based refolding additives, we focused on this tunable property and investigated the effects of new candidates such as N-alkylpyridinium chlorides and N-alkyl-N-methylpyrrolidinium chlorides on protein refolding. Denatured lysozyme (30 mg/mL) was used as a model protein and refolded by 30-fold dilution with various refolding buffers containing different ionic liquids consisting of a systematic variety of alkyl chains. Compared with the refolding yield without additives (lower than 10%), less hydrophobic ionic liquids such as N-ethyl, N-butyl and N-hexylpyridinium chlorides, and N-butyl-N-methylpyrrolidinium chloride were effective in enhancing the refolding yields (46-69%), because they primarily suppressed aggregation because of their chaotropic properties. N-alkylpyridinium cations were more hydrophobic than N-alkyl-N-methylpyrrolidinium cations according to the calculated log P values and prevented aggregation at lower concentrations because of their hydrophobicity. The results provide a range of new effective ionic liquid-based additives for higher protein refolding yields and the knowledge of the effect of chemical structures of additives on protein refolding.
    Applied biochemistry and biotechnology 02/2011; 164(6):957-67. · 1.94 Impact Factor
  • Satoshi Yamaguchi, Erika Matsunuma, Teruyuki Nagamune
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    ABSTRACT: Cell-based microarrays are promising tools for high-throughput functional analysis of gene products, but their application has been limited to adherent cells due to the difficulty in immobilization of non-adherent cells. Herein, we have introduced our techniques that can rapidly and strongly immobilize non-adherent cells and can allow the transfection of non-adherent cells with plasmid cDNA and small interfering RNA (siRNA) at a defined position on substrates with poly(ethylene-glycol)-lipid-modified surface.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 706:151-7. · 1.29 Impact Factor
  • Etsushi Yamamoto, Satoshi Yamaguchi, Teruyuki Nagamune
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    ABSTRACT: This paper presents the synergistic enhancement of the refolding yield of denatured and reduced lysozyme by using detergents as aggregation inhibitors and water-miscible organic cosolvents as modulators for the detergents. Adding only cetyltrimethylammonium bromide (CTAB) led to a slight increase in the refolding yield (up to 13%). Further addition of dimethylsulfoxide (DMSO) with CTAB drastically increased the refolding yield up to 35%, a value which was higher than the simple sum of the refolding yields in the presence of only CTAB or DMSO. The synergistic enhancement was also observed in the coexistence of other detergents, such as polyethylene glycol monooleyl ether (n = 50) and N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and cosolvents, such as N,N-dimethylformamide and N,N-dimethylacetamide. Experimental data and a kinetic analysis revealed the guideline for selecting a couple of additives; detergents which can adequately inhibit the aggregation of proteins by binding to hydrophobic surfaces of refolding intermediates should be employed as an aggregation inhibitor, and cosolvents which can properly prevent both protein-protein and protein-detergent interactions act as effective modulators for the aggregation inhibitor, resulting in a desirable balance between folding and aggregation rates.
    Journal of Bioscience and Bioengineering 01/2011; 111(1):10-5. · 1.74 Impact Factor

Publication Stats

287 Citations
127.24 Total Impact Points


  • 2001–2014
    • The University of Tokyo
      • • Department of Chemistry and Biotechnology
      • • Faculty & Graduate School of Engineering
      Edo, Tōkyō, Japan
  • 2013
    • Tokyo Institute of Technology
      • Chemical Resources Laboratory
      Tokyo, Tokyo-to, Japan
  • 2003
    • Nagasaki University Hospital
      Nagasaki, Nagasaki, Japan