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Engineering the Tobacco Etch Virus Protease toward a Platform for Traceless Cleavage Using Distal Site Prediction and
Martijn P Bemelmans1, Bach-Ngan Wetzel2, Florian G Neusius2
1SynBiofoundry@TUM, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany.
ACS Synthetic Biology
|August 23, 2025
Summary
Researchers engineered a new Tobacco Etch Virus protease (TEVp) variant, TEVp-C1, to enable traceless protein tag removal. This improved protease broadens substrate specificity, allowing cleavage after more amino acids for native peptide retrieval.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Engineering
Background:
- Protein tags are essential in biochemical engineering but require removal to avoid negatively impacting target molecules.
- Tobacco Etch Virus protease (TEVp) is commonly used for tag removal, but its strict P1' selectivity limits its application for noncanonical targets.
- Current TEVp cleavage leaves an N-terminal residue, preventing the recovery of native peptides.
Purpose of the Study:
- To engineer a novel TEVp variant with enhanced and broader substrate specificity for traceless protein tag cleavage.
- To overcome the limitations of native TEVp in cleaving noncanonical substrates and retrieving peptides with native N-termini.
Main Methods:
- Graph network analysis was employed to identify distal amino acid positions influencing TEVp activity.
- A smart library design approach was used to engineer TEVp variants.
- TEVp-C1 variant was created and tested against Switchtag-Teriparatide and fluorogenic peptide substrates.
- Mechanistic studies were performed to understand the functional impact of introduced mutations.
Main Results:
- The engineered TEVp-C1 variant demonstrated significantly improved cleavage for 15 of the 20 natural amino acids at the P1' position.
- TEVp-C1 showed enhanced activity against five disfavored residues at the P1' position.
- Mechanistic analysis indicated that mutations in TEVp-C1 facilitate the proton transfer step during catalysis.
- The engineered protease enables traceless cleavage, yielding peptides with native N-termini.
Conclusions:
- TEVp-C1 represents a promising protease platform for achieving traceless cleavage in protein engineering applications.
- The study validates the use of computational tools for predicting allosteric interactions to engineer enzyme substrate specificity.
- This work opens new avenues for customizing enzyme activity through targeted mutations at distal sites.
