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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

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Published on: January 16, 2019

A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled

Hidetaka Nakata¹, Takashi Ohtsuki, Masahiko Sisido

¹Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan.

Analytical Biochemistry

|April 28, 2009

Summary

We created new fluorescent protein substrates for measuring protease activity using fluorescence correlation spectroscopy (FCS). This novel method accurately detects protease activity and identifies potential inhibitors for drug discovery.

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Area of Science:

Biochemistry
Biophysics
Molecular Biology

Background:

Protease activity is crucial in biological processes, and its dysregulation is linked to various diseases.
Current methods for protease activity evaluation and inhibitor screening often have limitations in sensitivity or throughput.
Fluorescence Correlation Spectroscopy (FCS) offers a sensitive biophysical technique for analyzing molecular interactions and dynamics.

Purpose of the Study:

To develop novel, highly soluble, and sensitive substrates for protease activity assessment using FCS.
To validate the utility of these substrates for detecting specific protease activities, including caspase-3 and matrix metalloproteinase-9 (MMP-9).
To demonstrate the application of this FCS-based assay for high-throughput screening of protease inhibitors.

Main Methods:

Synthesized position-specific fluorescently labeled peptide substrates with a C-terminal elongation factor Ts (EF-Ts) protein tag.
Employed Fluorescence Correlation Spectroscopy (FCS) for sensitive detection of substrate cleavage and protease activity.
Screened a library of chemical compounds to identify inhibitors of MMP-9 using the developed FCS assay.

Main Results:

The novel substrates exhibited enhanced solubility and molecular weight, facilitating sensitive detection by FCS.
FCS successfully confirmed the activity of caspase-3 and MMP-9 using the developed substrates.
The high-throughput screening identified one known MMP-9 inhibitor and one potential novel inhibitor.

Conclusions:

The combination of position-specific labeled protein substrates and FCS provides a robust platform for protease activity evaluation.
This assay is suitable for sensitive detection of protease activity and effective for high-throughput screening of protease inhibitors.
This approach holds promise as a valuable tool in biochemical research and drug discovery efforts targeting proteases.