A novel bioluminescent protease assay using engineered firefly luciferase.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Proteases are crucial in disease pathogenesis, making them key targets for drug discovery.
• Understanding protease function is essential for developing new therapeutic strategies.

Purpose of the Study:

• To develop a novel, sensitive bioluminescent protease assay.
• To demonstrate the assay's utility in evaluating protease specificity and identifying inhibitors.

Main Methods:

• Engineered circularly permuted firefly luciferase with protease-cleavable sites.
• Assay development utilizing molecular cloning and cell-free translation for substrate generation.
• Application in high-throughput screening for protease inhibitor identification.

Main Results:

• Protease cleavage activated mutant luciferases over 100-fold.
• Demonstrated broad applicability across various proteases and recognition sites.
• Successfully utilized the assay for evaluating protease sequence specificity and inhibitor screening.

Conclusions:

• The developed bioluminescent assay offers a sensitive and versatile tool for protease research.
• Facilitates efficient identification and characterization of protease inhibitors for therapeutic development.