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 language | English |
|---|---|
| Pages (from-to) | 293-297 |
| Number of pages | 5 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 383 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Jun 5 2009 |
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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 journal › Article
}
TY - JOUR
T1 - A novel protease activity assay using a protease-responsive chaperone protein
AU - Sao, Kentaro
AU - Murata, Masaharu
AU - Fujisaki, Yuri
AU - Umezaki, Kaori
AU - Mori, Takeshi
AU - Niidome, Takuro
AU - Katayama, Yoshiki
AU - Hashizume, Makoto
PY - 2009/6/5
Y1 - 2009/6/5
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=67349240144&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2009.03.129
DO - 10.1016/j.bbrc.2009.03.129
M3 - Article
C2 - 19341711
AN - SCOPUS:67349240144
VL - 383
SP - 293
EP - 297
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -