Single-Walled Carbon Nanotube Probes for Protease Characterization Directly in Cell-Free Expression Reactions

Area of Science:

• Biotechnology
• Biochemistry
• Materials Science

Background:

• Cell-free expression (CFE) allows rapid protein prototyping.
• Limited availability of functional assays hinders high-throughput screening in CFE.
• Standardized data is crucial for machine learning-guided protein optimization.

Purpose of the Study:

• To develop novel fluorescent probes for direct protease activity measurement in cell-free lysate.
• To enhance the throughput of functional screens in cell-free protein expression systems.

Main Methods:

• Conjugating substrate proteins to carboxymethyl cellulose-wrapped single-walled carbon nanotubes (SWCNTs).
• Utilizing SWCNT probes for sensitive protease detection in complex cell lysate environments.
• Employing surface passivation techniques to improve probe stability and reduce signal loss.

Main Results:

• Developed stable and sensitive SWCNT-based protease probes with a detection limit of 6.4 ng/mL.
• Successfully measured subtilisin activity in unpurified CFE reactions, outperforming commercial assays.
• Enabled continuous monitoring of protease activity during protein synthesis in CFE systems without affecting expression.

Conclusions:

• Fluorescent SWCNT probes are effective tools for measuring protease activity directly in cell-free lysate.
• These modular probes facilitate high-throughput screening and optimization of proteases and other hydrolases.
• The developed probes advance the creation of standardized sequence-to-function datasets for protein engineering.