Teruo Akuta - Academia.edu (original) (raw)

Papers by Teruo Akuta

Research paper thumbnail of Sodium Dodecyl Sulfate Analogs as a Potential Molecular Biology Reagent

Current Issues in Molecular Biology, Jan 8, 2024

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Different behavior of Ferguson plot between agarose and polyacrylamide gels

Biophysical chemistry, Apr 1, 2024

Research paper thumbnail of Development of a novel two‐dimensional gel electrophoresis protocol with agarose native gel electrophoresis

Electrophoresis, Jun 9, 2023

A new protocol for conducting two‐dimensional (2D) electrophoresis was developed by combining the... more A new protocol for conducting two‐dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first‐dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high‐resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β‐galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Research paper thumbnail of Degradation of bradykinin by a metalloendopeptidase from Streptococcus pyogenes

Journal of Oral Biosciences, Nov 1, 2016

OBJECTIVES Streptococcus pyogenes secretes streptococcal pyrogenic exotoxin B (SpeB), which cleav... more OBJECTIVES Streptococcus pyogenes secretes streptococcal pyrogenic exotoxin B (SpeB), which cleaves kininogen to liberate bradykinin. In addition, this bacterium also has cell-associated bradykinin-degrading activity. Here, we characterized the bradykinin-degrading enzyme produced by S. pyogenes. METHODS The effects of various peptidase inhibitors on bradykinin degradation by intact S. pyogenes and cell lysates were assessed. Cleavage of bradykinin and other peptides by a recombinant putative metalloendopeptidase (Sp-Pep) from S. pyogenes was analyzed by mass spectrometry. The enhancement of vascular permeability induced by bradykinin (before and after treatment with Sp-Pep) was evaluated in guinea pig skin. RESULTS Various S. pyogenes strains expressed Sp-Pep. Immunoadsorption of S. pyogenes with an anti-Sp-Pep antibody showed that 80% of the bradykinin-degrading activity in S. pyogenes was due to Sp-Pep. Recombinant Sp-Pep cleaved bradykinin, and cleavage caused a loss of its extravasation-inducing potential. Sp-Pep-mediated degradation of bradykinin was 40 times more efficient than degradation of substance P and angiotensin II. While S. pyogenes secreted mature SpeB in stationary phase, this bacterium produced Sp-Pep during all tested growth phases. CONCLUSIONS S. pyogenes produces a cell-associated metalloendopeptidase that degrades bradykinin.

Research paper thumbnail of Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis

Research paper thumbnail of 一酸化窒素(NO)の新しいシグナル伝達機構 : グアノシンのニトロ化を介するNOシグナル

Research paper thumbnail of Western blotting analysis of proteins separated by agarose native gel electrophoresis

International Journal of Biological Macromolecules, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Elucidating the mechanisms of additive effects at high concentrations on hydrophobic interaction chromatography

Journal of Chromatography A

Research paper thumbnail of Ferguson Plot Analysis of Multiple Intermediate Species of Thermally Unfolded Bovine Serum Albumin

Research paper thumbnail of Agarose native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series

Research paper thumbnail of Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis

Research paper thumbnail of Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide

Protein expression and purification, Jan 25, 2015

We here present a new method for the expression and purification of recombinant human stem cell f... more We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accel...

Research paper thumbnail of Non-Affinity Purification of Antibodies

Antibodies

Currently, purification of antibodies is mainly carried out using a platform technology composed ... more Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Research paper thumbnail of Ladder observation of bovine serum albumin by high resolution agarose native gel electrophoresis

International Journal of Biological Macromolecules

Research paper thumbnail of Role of nitric oxide in IL-beta-induced chondrocyte death

Research paper thumbnail of Gel-electrophoresis based method for biomolecular interaction

Biomolecular Interactions Part B, 2022

Research paper thumbnail of A rabbit monoclonal antibody-mediated lateral flow immunoassay for rapid detection of CTX-M extended-spectrum β-lactamase-producing Enterobacterales

International Journal of Biological Macromolecules, 2021

Infections of CTX-M extended-spectrum β-lactamase-producing Enterobacterales are a severe threat ... more Infections of CTX-M extended-spectrum β-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A β-lactamases, including SHV and KPC. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test.

Research paper thumbnail of Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives

International Journal of Biological Macromolecules, 2022

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used... more Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

Research paper thumbnail of Extracellular vesicles from pancreatic ductal adenocarcinoma endoscopic ultrasound‐fine needle aspiration samples contain a protein barcode

Journal of Hepato-Biliary-Pancreatic Sciences, 2021

The survival rate of pancreatic ductal adenocarcinoma (PDAC) is very poor because early detection... more The survival rate of pancreatic ductal adenocarcinoma (PDAC) is very poor because early detection is difficult. Extracellular vesicles (EVs) are released from cells associating with the cellular condition and circulated in the blood. We aimed to identify EV proteins from endoscopic ultrasound‐fine needle aspiration (EUS‐FNA) biopsy samples in order to develop novel biomarkers for PDAC.

Research paper thumbnail of Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis

International Journal of Biological Macromolecules, 2021

Electrophoresis is one of the major techniques to analyze macromolecular structure and interactio... more Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

Research paper thumbnail of Sodium Dodecyl Sulfate Analogs as a Potential Molecular Biology Reagent

Current Issues in Molecular Biology, Jan 8, 2024

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Different behavior of Ferguson plot between agarose and polyacrylamide gels

Biophysical chemistry, Apr 1, 2024

Research paper thumbnail of Development of a novel two‐dimensional gel electrophoresis protocol with agarose native gel electrophoresis

Electrophoresis, Jun 9, 2023

A new protocol for conducting two‐dimensional (2D) electrophoresis was developed by combining the... more A new protocol for conducting two‐dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first‐dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high‐resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β‐galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Research paper thumbnail of Degradation of bradykinin by a metalloendopeptidase from Streptococcus pyogenes

Journal of Oral Biosciences, Nov 1, 2016

OBJECTIVES Streptococcus pyogenes secretes streptococcal pyrogenic exotoxin B (SpeB), which cleav... more OBJECTIVES Streptococcus pyogenes secretes streptococcal pyrogenic exotoxin B (SpeB), which cleaves kininogen to liberate bradykinin. In addition, this bacterium also has cell-associated bradykinin-degrading activity. Here, we characterized the bradykinin-degrading enzyme produced by S. pyogenes. METHODS The effects of various peptidase inhibitors on bradykinin degradation by intact S. pyogenes and cell lysates were assessed. Cleavage of bradykinin and other peptides by a recombinant putative metalloendopeptidase (Sp-Pep) from S. pyogenes was analyzed by mass spectrometry. The enhancement of vascular permeability induced by bradykinin (before and after treatment with Sp-Pep) was evaluated in guinea pig skin. RESULTS Various S. pyogenes strains expressed Sp-Pep. Immunoadsorption of S. pyogenes with an anti-Sp-Pep antibody showed that 80% of the bradykinin-degrading activity in S. pyogenes was due to Sp-Pep. Recombinant Sp-Pep cleaved bradykinin, and cleavage caused a loss of its extravasation-inducing potential. Sp-Pep-mediated degradation of bradykinin was 40 times more efficient than degradation of substance P and angiotensin II. While S. pyogenes secreted mature SpeB in stationary phase, this bacterium produced Sp-Pep during all tested growth phases. CONCLUSIONS S. pyogenes produces a cell-associated metalloendopeptidase that degrades bradykinin.

Research paper thumbnail of Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis

Research paper thumbnail of 一酸化窒素(NO)の新しいシグナル伝達機構 : グアノシンのニトロ化を介するNOシグナル

Research paper thumbnail of Western blotting analysis of proteins separated by agarose native gel electrophoresis

International Journal of Biological Macromolecules, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Elucidating the mechanisms of additive effects at high concentrations on hydrophobic interaction chromatography

Journal of Chromatography A

Research paper thumbnail of Ferguson Plot Analysis of Multiple Intermediate Species of Thermally Unfolded Bovine Serum Albumin

Research paper thumbnail of Agarose native gel electrophoresis analysis of thermal aggregation controlled by Hofmeister series

Research paper thumbnail of Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis

Research paper thumbnail of Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide

Protein expression and purification, Jan 25, 2015

We here present a new method for the expression and purification of recombinant human stem cell f... more We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accel...

Research paper thumbnail of Non-Affinity Purification of Antibodies

Antibodies

Currently, purification of antibodies is mainly carried out using a platform technology composed ... more Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Research paper thumbnail of Ladder observation of bovine serum albumin by high resolution agarose native gel electrophoresis

International Journal of Biological Macromolecules

Research paper thumbnail of Role of nitric oxide in IL-beta-induced chondrocyte death

Research paper thumbnail of Gel-electrophoresis based method for biomolecular interaction

Biomolecular Interactions Part B, 2022

Research paper thumbnail of A rabbit monoclonal antibody-mediated lateral flow immunoassay for rapid detection of CTX-M extended-spectrum β-lactamase-producing Enterobacterales

International Journal of Biological Macromolecules, 2021

Infections of CTX-M extended-spectrum β-lactamase-producing Enterobacterales are a severe threat ... more Infections of CTX-M extended-spectrum β-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A β-lactamases, including SHV and KPC. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test.

Research paper thumbnail of Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives

International Journal of Biological Macromolecules, 2022

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used... more Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

Research paper thumbnail of Extracellular vesicles from pancreatic ductal adenocarcinoma endoscopic ultrasound‐fine needle aspiration samples contain a protein barcode

Journal of Hepato-Biliary-Pancreatic Sciences, 2021

The survival rate of pancreatic ductal adenocarcinoma (PDAC) is very poor because early detection... more The survival rate of pancreatic ductal adenocarcinoma (PDAC) is very poor because early detection is difficult. Extracellular vesicles (EVs) are released from cells associating with the cellular condition and circulated in the blood. We aimed to identify EV proteins from endoscopic ultrasound‐fine needle aspiration (EUS‐FNA) biopsy samples in order to develop novel biomarkers for PDAC.

Research paper thumbnail of Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis

International Journal of Biological Macromolecules, 2021

Electrophoresis is one of the major techniques to analyze macromolecular structure and interactio... more Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.