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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
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.
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.
