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

作者：Sao Kentaro; Murata Masaharu^*; Fujisaki Yuri; Umezaki Kaori; Mori Takeshi; Niidome Takuro; Katayama Yoshiki; Hashizume Makoto

来源：Biochemical and Biophysical Research Communications, 2009, 383(3): 293-297.

DOI：10.1016/j.bbrc.2009.03.129

• Protease activity assay
• sHSP
• HSP16.5
• Chaperone-like activity
• Factor Xa protease
• Inhibitor

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 thi

• 出版日期2009-6-5