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Updated: Jul 23, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Engineering of TEV protease variants with redesigned substrate specificity.

Sebastian W Meister¹, Luke Parks¹, Leonie Kolmar¹

¹Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden.

Biotechnology Journal

|July 14, 2023

Summary

Researchers engineered proteases for new applications by redesigning their substrate specificity. A novel screening method successfully modified tobacco etch virus (TEV) protease to cleave a specific target peptide sequence.

Keywords:

GFP-fusion TEV protease amyloid betas intracellular assay protease engineering substrate specificity

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Area of Science:

Enzymology and Protein Engineering
Biotechnology and Industrial Applications

Background:

Proteases are enzymes that cleave proteins and peptides, offering potential in various industrial, biotechnological, and therapeutic fields.
Redesigning protease substrate specificity can significantly expand their practical applications.

Purpose of the Study:

To engineer tobacco etch virus (TEV) protease to recognize and cleave a novel target substrate (EKLVFQA).
To demonstrate a combinatorial screening method linking proteolytic activity to reporter protein folding and solubility.

Main Methods:

A semi-rational library of TEV protease variants was created with focused and random mutations.
Combinatorial screening coupled proteolytic activity with reporter protein solubility and correct folding.
Flow cytometry was used to sort and enrich protease variants with the desired activity.

Main Results:

The screening method successfully enriched protease variants capable of recognizing and cleaving the novel target substrate EKLVFQA.
The engineered variants demonstrated altered substrate specificity compared to the wild-type TEV protease.
The study validated a powerful approach for directed evolution of protease specificity.

Conclusions:

Engineered proteases with redesigned substrate specificities significantly broaden the scope of protease applications.
The developed combinatorial screening method is effective for directed evolution of enzyme specificity.
This work paves the way for custom-designed proteases for specific biotechnological and therapeutic needs.