Proteases

Proteases substrate specificity has many interesting applications in biology and human health. Previous work has demonstrated the ability to evolve proteases with altered specificity relevant to hepatitis C, botulinum neurotoxin, and systemic challenges such as pro-inflammatory cytokine IL-2. Drugs that inhibit activity of particular proteases in the body have long existed to target HIV, hepatitis C, and other infectious diseases.

We are developing a high-throughput measurement platform for protease function using a gene circuit that links protease activity to bacterial cell growth. The system is designed to measure the activity of proteases, particularly focusing on the Tobacco Etch Virus (TEV) protease as an initial target. The circuit relies on the cleavage of lysozyme from a T7 RNA polymerase, which then enables gene expression and antibiotic resistance.

The proposal details a two-stage approach for assay development, including tuning the system’s dynamic range and establishing normalization and calibration controls. The project aims to collect data on thousands of protease sequence variants against multiple substrates, with potential applications in understanding protease specificity and developing therapeutic interventions. Expansion of this dataset can include structural homologs of TEV protease and non-canonical substrates, which could have implications for drug development and understanding protease evolution.