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Updated: Apr 13, 2026

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Published on: April 6, 2016

PERRC: Protease Engineering with Reactant Residence Time Control.

Sage Nelson¹, Jokent Gaza², Seyednima Ajayebi¹

¹Department of Chemical Engineering, University of Florida, Gainesville 32611, United States.

ACS Synthetic Biology

|May 19, 2025

Summary

This summary is machine-generated.

Researchers engineered a new protease specificity platform, Protease Engineering with Reactant Residence Time Control (PERRC), to create highly specific proteases. This method enabled the development of an orthogonal protease variant with a 65-fold preference for a modified substrate.

Keywords:

high-throughput screening protease engineering synthetic biology tobacco etch virus (TEV) protease yeast surface display

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Last Updated: Apr 13, 2026

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

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Published on: April 6, 2016

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Published on: April 17, 2017

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

Area of Science:

Biotechnology
Molecular Biology
Enzyme Engineering

Background:

Proteases are crucial tools in biotechnology, but their broad substrate specificity limits applications.
Engineering precise protease specificity is challenging due to difficulties in counterselecting against native substrates.

Purpose of the Study:

To develop a novel platform for engineering protease specificity.
To create an orthogonal protease variant with enhanced substrate selectivity.

Main Methods:

Developed Protease Engineering with Reactant Residence Time Control (PERRC), a platform utilizing endoplasmic reticulum (ER) retention sequences to control protease evolution stringency.
Evolved an orthogonal tobacco etch virus protease variant using PERRC by adjusting selection and counterselection substrate ratios.

Main Results:

Successfully evolved TEVESNp, an orthogonal protease variant with a 65-fold preference for a substrate differing by a single amino acid from its parent sequence.
Demonstrated TEVESNp's efficacy in constructing orthogonal protein circuits in bacteria.
Molecular dynamics simulations revealed subtle active site rearrangements responsible for the enhanced specificity.

Conclusions:

PERRC is a modular system enabling precise engineering of protease specificity.
The developed orthogonal protease variant has significant potential for targeted therapeutics and biotechnology.
This work represents a breakthrough in engineering highly specific proteases from subtle sequence differences.