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

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

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)293-297
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume383
Issue number3
DOIs
Publication statusPublished - Jun 5 2009

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Assays
Peptide Hydrolases
Proteins
Factor Xa
Small Heat-Shock Proteins
Nephelometry and Turbidimetry
Recombinant Proteins
Radioisotopes
Labeling
Bacteria
Agglomeration
Temperature
Therapeutics

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A novel protease activity assay using a protease-responsive chaperone protein. / Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto.

In: Biochemical and Biophysical Research Communications, Vol. 383, No. 3, 05.06.2009, p. 293-297.

Research output: Contribution to journalArticle

Sao, Kentaro ; Murata, Masaharu ; Fujisaki, Yuri ; Umezaki, Kaori ; Mori, Takeshi ; Niidome, Takuro ; Katayama, Yoshiki ; Hashizume, Makoto. / A novel protease activity assay using a protease-responsive chaperone protein. In: Biochemical and Biophysical Research Communications. 2009 ; Vol. 383, No. 3. pp. 293-297.
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