Higeta Shoyu Co.

Chiba, Japan

Higeta Shoyu Co.

Chiba, Japan
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Nagai H.,Kyoto University | Ebisu S.,Protein Express Co. | Ebisu S.,HIGETA SHOYU Co. | Abe R.,Protein Express Co. | And 5 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2011

The activation of peroxisome-proliferator-activated receptor-γ (PPARγ), which plays a central role in adipocyte differentiation, depends on ligand-dependent co-activator recruitment. In this study, we developed a novel method of PPARγ ligand screening by measuring the increase in fluorescent polarization accompanied by the interaction of a fluorescent co-activator and PPARγ. Sterol receptor co-activator-1 (SRC-1), a major PPARγ co-activator, was probed by fluorescent TAMRA by the Amber codon fluorescence probe method. Polarization was increased by adding PPARγ ligands to a solution containing labeled SRC-1 (designated TAMRA-SRC-S) and PPARγ. The disassociation constants (Kd) of the PPARγ synthesized ligands, pioglitazone (221 nM), tro-glitazone (83.0 nM), and 15-deoxy-Δ12,14-prostaglandin J2 (15d-ΔPGJ2) (156 nM), were determined by this method. Farnesol (2.89 μm) and bixin (21.1 μm), which we have reported to be PPARγ ligands, increased the fluorescent polarization. Their Kd values were in agreement with the ED 50 values obtained in the luciferase assay. The results indicate that the method is valuable for screening natural PPARγ ligands.


Kawaki H.,Okayama University of Science | Kubota S.,Okayama University of Science | Aoyama E.,Okayama University | Fujita N.,Tokyo Medical University | And 6 more authors.
Biochimie | Year: 2010

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) consists of 4 conserved modules that are highly interactive with a number of biomolecules. With such interaction, CCN2 exerts multiple functions by forming an extracellular information network. In the present study, we screened for dodecapeptide sequences that bound to each module of human CCN2 by using a bacteriophage display library. Thereafter, consensus amino acid sequences for the binding to individual modules were extracted in silico and utilized to design anchor peptide aptamers that would facilitate the interaction between CCN2 and other molecules. Direct binding of a few peptides to CCN2 was confirmed by surface plasmon resonance analysis. Subsequent biological assay indicated that one such peptide was capable of promoting the proliferation of CCN2-producing chondrocytic cells. This cell biological activity was found to be sequence specific and CCN2 dependent. Since CCN2/CTGF was shown to be effective in articular cartilage/bone regeneration in vivo, utility of such peptide aptamers in CCN2-associated regenerative therapeutics is suggested herein. © 2010 Elsevier Masson SAS.


Mizukami M.,Higeta Shoyu Co. | Tokunaga H.,Kagoshima University | Onishi H.,Higeta Shoyu Co. | Ueno Y.,Higeta Shoyu Co. | And 9 more authors.
Protein Expression and Purification | Year: 2015

Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95 mg in 100 ml conventional flask culture. With a 3-L scale fed-batch culture for 65 h, the N15 VHH protein with C-terminal His-tag was produced at ∼3 g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD = 6.0-8.6 nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232 nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67 °C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation. © 2014 Elsevier Inc. All rights reserved.


Tokunaga M.,Kagoshima University | Mizukami M.,Higeta Shoyu Co. | Yamasaki K.,Kagoshima University | Tokunaga H.,Kagoshima University | And 6 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution. © 2013 Springer-Verlag Berlin Heidelberg.


Mizukami M.,Higeta Shoyu Co. | Hanagata H.,Higeta Shoyu Co. | Miyauchi A.,Higeta Shoyu Co.
Current Pharmaceutical Biotechnology | Year: 2010

Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (-30g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants. © 2010 Bentham Science Publishers Ltd.


Onishi H.,Higeta Shoyu Co. | Mizukami M.,Higeta Shoyu Co. | Hanagata H.,Higeta Shoyu Co. | Tokunaga M.,Kagoshima University | And 2 more authors.
Protein Expression and Purification | Year: 2013

Expression of scFv in Brevibacillus choshinensis was tested using combinations of three different promoters and four different secretion signals. Two model scFv constructs, i.e., His-scFvFLU and His-scFvHEL, were successfully expressed with some of the combinations. Ni Sepharose column and size exclusion chromatography resulted in fairly pure preparations of these two proteins. The purified His-scFvFLU inhibited fluorescence from fluorescein, while the purified His-scFvHEL inhibited lysozyme activity. Relatively high yield of His-scFvFLU (∼40%) and His-scFvHEL (∼30%) was achieved with the expression and purification system described here.© 2013 Published by Elsevier Inc.


PubMed | Japan National Institute of Advanced Industrial Science and Technology, Alliance Protein Laboratories, Kagoshima University and Higeta Shoyu Co.
Type: | Journal: Protein expression and purification | Year: 2016

Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at 3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.


PubMed | Higeta Shoyu Co.
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2016

Brevibacillus choshinensis is an excellent host for the production of secretory proteins. This host has also been applied successfully to efficient production of CCN proteins. Described herein are methods of constructing plasmids for CCN protein production (IGFBP-, VWC-, TSP-, and CT-domain) with Brevibacillus as a host, cultivation methods for protein production, and methods of purification for domain proteins using his-tag.

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