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

• 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
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2009.03.129

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.