Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protease activity monitoring is crucial in various biological and biotechnological applications.
• Existing methods for monitoring protease activity can be limited in dynamic range or sensitivity.
• PreScission Protease (PSP) is widely used for protein purification and requires accurate activity assessment.

Purpose of the Study:

• To develop a novel, sensitive, and dynamic assay for monitoring PreScission Protease (His-PSP) activity.
• To utilize Förster Resonance Energy Transfer (FRET) between quantum dots (QDs) and a fluorescently labeled peptide substrate.
• To combine capillary electrophoresis with fluorescence detection (CE-FL) for real-time cleavage monitoring.

Main Methods:

• Construction of a multivalent peptide substrate by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto CdSe/ZnS quantum dots (QDs).
• Utilizing the FRET phenomenon from QDs to ATTO 590 in the immobilized substrate.
• Incubating the ATTO-LEVH6-QD assembly with His-PSP and analyzing the proteolytic cleavage products using CE-FL.

Main Results:

• Demonstrated successful immobilization of the peptide substrate onto QDs, enabling FRET.
• Showcased the dynamic monitoring of His-PSP mediated cleavage of the surface-bound substrate using CE-FL.
• Established that PreScission-mediated proteolytic cleavage is dependent on enzyme concentration and incubation time.

Conclusions:

• A novel CE-FL based assay was successfully developed for the dynamic monitoring of PreScission Protease activity.
• The assay leverages QDs and FRET for sensitive detection of enzyme cleavage.
• This method offers a versatile platform that can be extended for the detection and monitoring of various other enzymes and proteases.