PERRC: Protease Engineering with Reactant Residence Time Control

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.