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

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat...

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Published in	Biochemical and biophysical research communications Vol. 383; no. 3; pp. 293 - 297
Main Authors	Sao, Kentaro, Murata, Masaharu, Fujisaki, Yuri, Umezaki, Kaori, Mori, Takeshi, Niidome, Takuro, Katayama, Yoshiki, Hashizume, Makoto
Format	Journal Article
Language	English
Published	United States Elsevier Inc 05.06.2009
Subjects	60 APPLIED LIFE SCIENCES AGGLOMERATION BACTERIA Chaperone-like activity CONCENTRATION RATIO ECOLOGICAL CONCENTRATION ENVIRONMENTAL SCIENCES Factor Xa - genetics Factor Xa - metabolism Factor Xa protease HEAT-SHOCK PROTEINS Heat-Shock Proteins, Small - genetics Heat-Shock Proteins, Small - metabolism HSP16.5 Inhibitor Molecular Chaperones - genetics Molecular Chaperones - metabolism Nephelometry and Turbidimetry - methods Peptide Hydrolases - analysis Peptide Hydrolases - metabolism PRECIPITATION Protease activity assay RADIOISOTOPES Recombinant Proteins - genetics Recombinant Proteins - metabolism sHSP TEMPERATURE RANGE 0400-1000 K HSP16.5 Chaperone-like activity Protease activity assay Factor Xa protease Inhibitor sHSP
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Abstract	Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.
AbstractList	Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.
Author	Mori, Takeshi Hashizume, Makoto Murata, Masaharu Niidome, Takuro Sao, Kentaro Katayama, Yoshiki Fujisaki, Yuri Umezaki, Kaori
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CitedBy_id	crossref_primary_10_3390_bios11040117 crossref_primary_10_1016_j_pep_2014_12_001 crossref_primary_10_3748_wjg_v21_i24_7400 crossref_primary_10_1021_acsami_5b11902 crossref_primary_10_1039_c3cc44508a crossref_primary_10_1021_mp5007129 crossref_primary_10_1021_bc300015f
Cites_doi	10.1016/j.bbrc.2004.04.098 10.1016/j.bmc.2008.11.013 10.1074/jbc.M311710200 10.1006/bbrc.1997.6079 10.1038/nrd2092 10.1074/jbc.M608322200 10.1006/abio.1995.1264 10.1021/ja057499n 10.1126/science.2106161 10.1074/jbc.275.6.3767 10.1039/b709775a 10.1016/S0006-291X(03)01302-0 10.1038/nrc2228 10.1002/ajh.21220 10.1007/s00018-006-6321-2 10.1073/pnas.95.16.9129 10.1073/pnas.0510770103 10.1073/pnas.1032940100 10.1038/29106 10.1038/nsmb993 10.1006/bbrc.2001.5921
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Snippet	Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method...
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SubjectTerms	60 APPLIED LIFE SCIENCES AGGLOMERATION BACTERIA Chaperone-like activity CONCENTRATION RATIO ECOLOGICAL CONCENTRATION ENVIRONMENTAL SCIENCES Factor Xa - genetics Factor Xa - metabolism Factor Xa protease HEAT-SHOCK PROTEINS Heat-Shock Proteins, Small - genetics Heat-Shock Proteins, Small - metabolism HSP16.5 Inhibitor Molecular Chaperones - genetics Molecular Chaperones - metabolism Nephelometry and Turbidimetry - methods Peptide Hydrolases - analysis Peptide Hydrolases - metabolism PRECIPITATION Protease activity assay RADIOISOTOPES Recombinant Proteins - genetics Recombinant Proteins - metabolism sHSP TEMPERATURE RANGE 0400-1000 K
Title	A novel protease activity assay using a protease-responsive chaperone protein
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