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The Proteasome

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...

The Proteasome

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...

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Related Experiment Video

Updated: Jan 17, 2026

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

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

Published on: January 16, 2019

High-Throughput Activity Reprogramming of Proteases (HARP).

Samantha G Martinusen¹, Ethan W Slaton¹, Seyednima Ajayebi¹

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

ACS Chemical Biology

|September 23, 2025

Summary

This summary is machine-generated.

The High-throughput Activity Reprogramming of Proteases (HARP) platform efficiently discovers potent protease inhibitors. This yeast-based screen isolates inhibitory macromolecules, enabling low-nanomolar inhibitor discovery for enzymes like TEVp.

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Area of Science:

Biochemistry
Molecular Biology
Enzyme Inhibitor Discovery

Background:

Developing selective protease inhibitors is challenging due to the difficulty in isolating rare, potent molecules.
Existing platforms prioritize high-affinity binders, necessitating extensive downstream characterization.

Purpose of the Study:

To develop a novel, high-throughput platform for discovering protease-inhibitory macromolecules.
To isolate potent and selective inhibitors against specific protease targets.

Main Methods:

Developed the High-throughput Activity Reprogramming of Proteases (HARP) platform, a yeast-based functional screen.
Coupled protease inhibition to a selectable cell surface phenotype for efficient screening of large libraries.
Utilized structural modeling and deep sequencing for inhibitor analysis.

Main Results:

Successfully isolated low-nanomolar inhibitory nanobodies against tobacco etch virus protease and human kallikrein 6.
Identified a rare 10.5 nM K_I uncompetitive inhibitor for TEVp.
HARP demonstrated high dynamic range and resolution in inhibitor discovery.

Conclusions:

HARP is a premier platform for discovering modulatory macromolecules against enzyme targets.
The platform facilitates the isolation of potent inhibitors from diverse synthetic scaffolds.
Insights into molecular determinants of inhibition were gained through structural modeling and deep sequencing.