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Related Experiment Video

Updated: Feb 14, 2026

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Published on: January 29, 2018

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity.

Wan-Shan Yang¹, Mel Campbell², Hsing-Jien Kung³

¹Institute of Microbiology and Immunology, National Yang-Ming University.

Journal of Visualized Experiments : Jove

|February 15, 2018

Summary

This study presents a refined in vitro SUMOylation protocol to identify SUMO E3 ligase activity and presents a method to purify K-bZIP, a viral SUMO-2/3 E3 ligase, demonstrating its SUMOylation of p53.

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Last Updated: Feb 14, 2026

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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Published on: January 29, 2018

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Published on: May 14, 2021

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

Area of Science:

Molecular Biology
Post-Translational Modifications
Biochemistry

Background:

Small ubiquitin-like modifier (SUMO)ylation is a crucial post-translational modification regulating protein interactions and signal transduction.
SUMOylation involves an enzymatic cascade: E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3 ligases, with E3 ligases enhancing specificity and efficiency.
Detecting E3 ligase activity in vitro is challenging as E1 and E2 enzymes alone can mediate SUMO conjugation.

Purpose of the Study:

To develop a robust modified in vitro SUMOylation protocol for consistently identifying SUMO modification.
To present a protocol for purifying catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase.
To demonstrate the utility of the protocol for discovering novel SUMO E3 ligases and their paralog specificity.

Main Methods:

Development of a modified in vitro SUMOylation assay using purified components.
Step-by-step protocol for the purification of catalytically active K-bZIP E3 ligase.
In vitro SUMOylation assay demonstrating K-bZIP's activity on p53.

Main Results:

The modified in vitro SUMOylation protocol reliably identifies SUMO modification in a reconstituted system.
Catalytically active K-bZIP was successfully purified.
Purified K-bZIP demonstrated SUMO-2/3 E3 ligase activity, SUMOylating the known target p53.

Conclusions:

The developed protocol provides a consistent method for identifying SUMO E3 ligase activity in vitro.
This method facilitates the discovery of novel SUMO E3 ligases and characterization of their substrate and paralog specificity.
The purification and characterization of K-bZIP highlight its utility as a tool for studying SUMOylation.