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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Assays

Background:

• Protease activity assays are crucial for understanding enzyme function and developing therapeutics.
• Existing methods often require labeling or radioisotopes, limiting accessibility and increasing complexity.

Purpose of the Study:

• To develop a novel, label-free turbidimetric assay for quantifying protease activity.
• To utilize a protease-responsive chaperone protein for a simple and effective detection method.

Main Methods:

• Synthesized a recombinant small heat-shock protein (sHSP) with a Factor Xa protease recognition site (FXa-HSP).
• Investigated the chaperone-like activity of FXa-HSP at high temperatures in cell lysates.
• Assessed the effect of Factor Xa treatment on FXa-HSP activity and subsequent protein precipitation.
• Quantified protease activity using turbidimetry to detect protein aggregation.

Main Results:

• FXa-HSP exhibited temperature-dependent chaperone activity that was significantly reduced upon Factor Xa cleavage.
• Factor Xa treatment led to concentration-dependent protein precipitation, detectable by turbidimetry.
• The assay's effectiveness was confirmed by its quantitative suppression using a specific Factor Xa inhibitor.
• Protein aggregation correlated directly with protease activity.

Conclusions:

• The developed turbidimetric assay provides an effective and user-friendly method for determining protease activity.
• This novel approach eliminates the need for labeling procedures and radioisotopes.
• The assay is suitable for various applications, including drug discovery and basic research.