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Related Experiment Video

Updated: Jun 13, 2025

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Published on: June 20, 2019

Engineering high-efficiency matriptase substrates using E. coli display for applications in prodrug activation.

Anna Mestre Borras¹, Hanna Mehari¹, Stefan Ståhl¹

¹Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden.

Cell Reports Methods

|June 11, 2025

Summary

This summary is machine-generated.

Researchers engineered new protease substrates for matriptase, a tumor-associated enzyme. This breakthrough enhances tumor-targeted therapies using protease-activated prodrugs for greater treatment selectivity.

Keywords:

CP: Biotechnology CP: Molecular biology E. coli display antibody engineering conditional activation directed evolution matriptase prodrug protease substrate protein engineering tumor protease

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Last Updated: Jun 13, 2025

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

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Published on: June 20, 2019

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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Published on: January 16, 2019

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Published on: October 3, 2019

Area of Science:

Biochemistry
Molecular Biology
Oncology

Background:

Proteases are vital for biological processes, including tumor growth and tissue balance.
Protease-activated prodrugs offer enhanced selectivity for tumor-targeted therapies.
Matriptase, a protease overexpressed in tumors, is a target for prodrug activation.

Purpose of the Study:

To engineer improved substrate sequences for matriptase.
To enhance the efficiency of prodrug activation for cancer therapies.

Main Methods:

A peptide library with millions of potential substrates was displayed on Escherichia coli.
Flow cytometry was used for sorting and isolating substrates based on cleavage efficiency.
The top substrates were evaluated in an antibody-prodrug system.

Main Results:

Engineered matriptase substrates showed over 40-fold higher catalytic efficiency (kcat/KM) compared to previous sequences.
These substrates demonstrated exceptional activation in an antibody-prodrug format.
The bacterial display platform proved effective for substrate discovery.

Conclusions:

The engineered matriptase substrates have significant potential for applications like cleavable linkers in advanced antibody prodrugs.
The bacterial display platform is a promising tool for discovering substrates for other proteases.
This work advances the development of targeted cancer therapies.