title: A novel protease activity assay using a protease-responsive chaperone protein.
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.
authors.affiliation: Kyushu University, Motooka Nishi-ku, Fukuoka, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Sao
authors.forename: Masaharu
authors.initials: M
authors.lastname: Murata
authors.forename: Yuri
authors.initials: Y
authors.lastname: Fujisaki
authors.forename: Kaori
authors.initials: K
authors.lastname: Umezaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mori
authors.forename: Takuro
authors.initials: T
authors.lastname: Niidome
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Katayama
authors.forename: Makoto
authors.initials: M
authors.lastname: Hashizume