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Single-Walled Carbon Nanotube Probes for Protease Characterization Directly in Cell-Free Expression Reactions
Sepehr Hejazi1, Ryan Godin1, Vito Jurasic1
1Chemical and Biological Engineering, Iowa State University, 618 Bissell Road, Ames, Iowa 50011, United States.
Analytical Chemistry
|May 14, 2025
Summary
Researchers developed fluorescent probes using single-walled carbon nanotubes (SWCNT) to measure protease activity in cell-free systems. This breakthrough enables high-throughput screening for protein optimization using machine learning.
Area of Science:
- Biochemistry and Molecular Biology
- Materials Science
- Biotechnology
Background:
- Cell-free expression (CFE) allows rapid protein prototyping but lacks functional assays in cell lysate, limiting screening throughput.
- High-throughput functional data is crucial for machine learning-guided protein optimization and standardized datasets.
- Existing assays are often unsuitable for direct use in complex, unpurified CFE reactions.
Purpose of the Study:
- To develop novel fluorescent probes for direct measurement of protease activity in cell-free expression (CFE) lysates.
- To enable high-throughput screening and continuous monitoring of protease activity within CFE systems.
- To create modular probes adaptable for various hydrolase activities.
Main Methods:
- Conjugation of substrate proteins to carboxymethyl cellulose-wrapped single-walled carbon nanotubes (SWCNT) to create fluorescent probes.
- Testing probe sensitivity and detection limits using bacterial protease from *Streptomyces griseus*.
- Application of probes in unpurified CFE reactions and continuous monitoring during protein synthesis.
- Implementation of surface passivation techniques to enhance probe stability in complex lysate environments.
Main Results:
- Developed stable and sensitive SWCNT-based probes for protease detection with a low detection limit (6.4 ng/mL for bacterial protease).
- Successfully measured subtilisin activity in unpurified CFE reactions, outperforming commercial assays.
- Enabled continuous monitoring of protease activity during synthesis in both purified and lysate-based CFE systems without inhibiting expression.
- Surface passivation improved probe signal stability and reduced initial signal loss in complex cell lysates.
Conclusions:
- Fluorescent SWCNT probes provide a robust and sensitive method for measuring protease activity directly in CFE lysates.
- These probes facilitate high-throughput screening and continuous monitoring, advancing protein engineering and optimization.
- The modular nature of the probes allows for adaptation to assay a wider range of hydrolases.
