title: Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli.
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.
authors: Department 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.
authors: Elvin D
authors: ED
authors: de Araujo
authors: Department 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.
authors: Mulu
authors: M
authors: Geletu
authors: Department 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. Electronic address: patrick.gunning@utoronto.ca.
authors: Patrick T
authors: PT
authors: Gunning
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D050799:STAT5 Transcription Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human
keywords: Osmolyte; Peptide fluorescence polarization; Recombinant Signal Transducer and Activator of Transcription 5B (STAT5B) expression; SYPRO Orange; Thermal shift assay; Western blotting