A novel protease activity assay using a protease-responsive chaperone protein

Sao, Kentaro; Murata, Masaru; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

doi:10.1016/j.bbrc.2009.03.129

Cited by 10 publications

(7 citation statements)

References 21 publications

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“…The outer and inner diameters of the cage are 13 and 6.5 nm, respectively. − As stable cage-like biomolecules that circulate in the blood, HSP-based nanomaterials possess good biocompatibility and adequate stability. We recently reported the design, synthesis, and biological characterization of several types of protein nanoprobes. − HSP nanoprobes are attractive as a biomedical tool for the delivery of imaging agents because of their biocompatibility, monodispersed formation, robust structure, easy acquisition from Escherichia coli ( E. coli ), and simple functionalization through chemical and genetic strategies.…”

Section: Introductionmentioning

confidence: 99%

Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula

Hamano

Murata

Kawano

et al. 2016

ACS Appl. Mater. Interfaces

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Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastroenterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor-acceptor dye pair for Förster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, α-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.

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Section: Introductionmentioning

confidence: 99%

Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula

Hamano

Murata

Kawano

et al. 2016

ACS Appl. Mater. Interfaces

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“…[13][14][15][16] These nanocapsules are self-organized protein oligomer complexes with nanoscale inner cavities and whose three-dimensional architecture can be specified in great detail. 17,18 Thus, they have the potential to offer special advantages in developing new functions that are responsive to biochemical signals exhibited by various cellular processes.…”

Section: Introductionmentioning

confidence: 99%

Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages

Murata¹,

Narahara²,

Umezaki³

et al. 2012

IJN

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Abstract:Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.

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“…However, spectroscopic assays are incapable of measuring protease activity in highly colored and turbid samples such as cells, tissue lysates, or milk. Therefore, the development of a new label-free method for detecting protease activity without interruption from impurity inclusions is needed [1,15,17].…”

Section: Introductionmentioning

confidence: 99%

Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry

et al. 2021

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The determination of protease activity is very important for disease diagnosis, drug development, and quality and safety assurance for dairy products. Therefore, the development of low-cost and sensitive methods for assessing protease activity is crucial. We report two approaches for monitoring protease activity: in a volume and at surface, via colorimetric and acoustic wave-based biosensors operated in the thickness-shear mode (TSM), respectively. The TSM sensor was based on a β-casein substrate immobilized on a piezoelectric quartz crystal transducer. After an enzymatic reaction with trypsin, it cleaved the surface-bound β-casein, which increased the resonant frequency of the crystal. The limit of detection (LOD) was 0.48 ± 0.08 nM. A label-free colorimetric assay for trypsin detection has also been performed using β-casein and 6-mercaptohexanol (MCH) functionalized gold nanoparticles (AuNPs/MCH-β-casein). Due to the trypsin cleavage of β-casein, the gold nanoparticles lost shelter, and MCH increased the attractive force between the modified AuNPs. Consequently, AuNPs aggregated, and the red shift of the absorption spectra was observed. Spectrophotometric assay enabled an LOD of 0.42 ± 0.03 nM. The Michaelis–Menten constant, KM, for reverse enzyme reaction has also been estimated by both methods. This value for the colorimetric assay (0.56 ± 0.10 nM) is lower in comparison with those for the TSM sensor (0.92 ± 0.44 nM). This is likely due to the better access of the trypsin to the β-casein substrate at the surface of AuNPs in comparison with those at the TSM transducer.

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A novel protease activity assay using a protease-responsive chaperone protein

Cited by 10 publications

References 21 publications

Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula

Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula

Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages

Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry

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