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Published on: October 4, 2017
Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli
Elvin D de Araujo1, Mulu Geletu1, Patrick T Gunning1
1Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada.
Abstract:
STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.
Insights
Researchers optimized recombinant STAT5B protein production in E. coli for cancer drug screening. This improved yield and purity of STAT5B protein, crucial for developing new cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Signal transducer and activator of transcription 5B (STAT5B) is a transcription factor involved in cancer development.
- Inhibiting STAT activity presents therapeutic opportunities for cancer treatment.
- High-throughput biophysical screening requires efficient production of high-purity, full-length STAT proteins.
Purpose of the Study:
- To optimize recombinant STAT5B protein expression and purification from E. coli.
- To establish a cost-effective method for producing functional STAT5B protein for drug screening.
Main Methods:
- Exploration of diverse protein expression and purification conditions.
- Recombinant protein expression in E. coli.
- Western blotting for protein identity confirmation.
- Fluorescence polarization and thermal shift assays for protein functionality and stability assessment.
Main Results:
- Substantially increased yields of recombinant STAT5B protein.
- Confirmed protein identity and functionality through biochemical assays.
- Identified optimal expression conditions for efficient recovery of soluble STAT5B protein.
Conclusions:
- The developed method significantly enhances the yield and purity of STAT5B protein.
- This facilitates biophysical studies and high-throughput screening for STAT5B-targeting cancer drugs.
- The optimized production is key for advancing STAT5B-focused therapeutic strategies.
