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Recombinant Topoisomerase 2 Production Using Cultured Human Cell Lines.

Anh T Q Cong¹, Matthew J Schellenberg²

¹Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.

Methods in Molecular Biology (Clifton, N.J.)

|May 15, 2025

Summary

This summary is machine-generated.

We developed a scalable method for producing high-purity recombinant human Topoisomerase 2 (TOP2) alpha and beta proteins. This method uses HEK293F cells and fluorescent protein fusions for efficient purification and structural analysis.

Keywords:

Nanobody Recombinant protein expression Topoisomerase 2 Transfection YFP-tag mCherry-tag

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Area of Science:

Biochemistry
Molecular Biology
Protein Expression

Background:

Recombinant human Topoisomerase 2 (TOP2) alpha and beta are crucial for structural and biochemical studies.
Existing methods may not provide sufficient purity or yield for these analyses.

Purpose of the Study:

To develop a scalable and efficient method for producing high-purity recombinant human TOP2 alpha and beta proteins.
To enable structural and biochemical investigations of TOP2 enzymes.

Main Methods:

Utilized a HEK293F cell expression system with transient transfection.
Expressed TOP2 as a fusion protein with YFP or mCherry for expression monitoring and affinity purification.
Employed Fast Protein Liquid Chromatography (FPLC) for protein polishing.

Main Results:

Achieved milligram-scale yields of high-purity recombinant TOP2 proteins.
Demonstrated the activity of the purified recombinant TOP2.
The YFP/mCherry fusion facilitated rapid isolation and purification.

Conclusions:

The described method provides a scalable and robust approach for producing active, high-purity recombinant human TOP2.
This facilitates further structural and biochemical research on TOP2 enzymes.