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

Kentaro Sao, Masaharu Murata, Yuri Fujisaki, Kaori Umezaki, Takeshi Mori, Takuro Niidome, Yoshiki Katayama, Makoto Hashizume

研究成果: Contribution to journalArticle査読

9 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)293-297
ページ数5
ジャーナルBiochemical and Biophysical Research Communications
383
3
DOI
出版ステータス出版済み - 6 5 2009

All Science Journal Classification (ASJC) codes

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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