A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Proteases play crucial roles in biological processes.
• Engineering and characterizing protease activity and specificity are vital for research and industry.

Purpose of the Study:

• To present a novel method for assessing site-specific proteolysis.
• To facilitate protease substrate identification and directed evolution.

Main Methods:

• Utilizing plasmid-encoded reporters that confer antibiotic resistance upon protease cleavage.
• Co-expressing reporters with proteases (e.g., tobacco etch virus protease - TEVp) and selecting in antibiotic-containing media.
• Employing competitive growth assays to enrich for efficient protease substrates.

Main Results:

• Cells with cleavable reporters showed antibiotic resistance, correlating with cleavage efficiency.
• TEVp-mediated cleavage was successfully assessed using the antibiotic resistance assay.
• Competitive growth enriched preferred TEVp substrates over less efficient ones.

Conclusions:

• The developed assay provides a simple and effective method for assessing site-specific proteolysis.
• This methodology holds significant promise for protease substrate identification and directed evolution.
• The approach can be used to engineer proteases and recognition sequences for improved or novel functions.