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-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.
Sao
Kentaro
K
Kyushu University, Motooka Nishi-ku, Fukuoka, Japan.
Murata
Masaharu
M
Fujisaki
Yuri
Y
Umezaki
Kaori
K
Mori
Takeshi
T
Niidome
Takuro
T
Katayama
Yoshiki
Y
Hashizume
Makoto
M