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Updated: Jun 10, 2025
Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells
Published on: July 30, 2020
Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner
Jonna Mattsson1, Per Rogne1, Maréne Landström2
1Department of Chemistry, Umeå University, 901 87, Umeå, Sweden.
Abstract:
Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H-15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.
Insights
Researchers developed a novel E. coli system to produce milligram quantities of active human Aurora B kinase complexed with INCENP. This breakthrough facilitates crucial structural and functional studies for developing new cancer drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Protein kinases, including Aurora kinases, are crucial in cell signaling and implicated in human cancers.
- Human Aurora kinase B has been difficult to study due to challenges in producing sufficient quantities of active enzyme.
Purpose of the Study:
- To develop a novel E. coli-based system for producing large quantities of active human Aurora B:INCENP complex.
- To enable structural and functional studies of Aurora B for potential anticancer drug development.
Main Methods:
- Engineered E. coli expression system for producing a continuous polypeptide chain of Aurora B and INCENP.
- TEV protease cleavage to generate a stable heterodimer.
- Activity, stability, and phosphorylation assessed using ATPase assays, 31P NMR, and mass spectrometry.
- Isotope labeling enabled for 1H-15N-HSQC analysis.
Main Results:
- Successfully produced milligram quantities of well-folded, active human Aurora B:INCENP complex.
- Characterized the complex's activity, stability, and phosphorylation status.
- Generated the first 1H-15N-HSQC spectrum of the human Aurora B:INCENP complex, confirming successful isotope labeling.
Conclusions:
- The developed E. coli production system overcomes previous limitations in studying human Aurora B.
- This strategy provides a foundation for future structural and functional investigations of Aurora B.
- Facilitates the development of targeted anticancer therapies by enabling detailed study of this key mitotic protein kinase.
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