Recombinant EWS-FLI1 Oncoprotein Activates Transcription

• Published in: Biochemistry (Easton) 2004-10, Vol.43 (42), p.13579-13589
• Main Authors: Üren, Aykut, Tcherkasskaya, Olga, Toretsky, Jeffrey A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Circular Dichroism; Cloning, Molecular; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; Escherichia coli; Escherichia coli - genetics; Humans; Molecular Sequence Data; Oncogene Proteins, Fusion - biosynthesis; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - physiology; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Protein c-fli-1; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - physiology; RNA-Binding Protein EWS - chemistry; RNA-Binding Protein EWS - genetics; Sarcoma, Ewing - chemistry; Sarcoma, Ewing - drug therapy; Sarcoma, Ewing - genetics; Spectrometry, Fluorescence; Spodoptera - genetics; Trans-Activators - biosynthesis; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - physiology; Transcription Factors - biosynthesis; Transcription Factors - genetics; Transcription Factors - physiology; Transcriptional Activation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048776q

The Ewing's sarcoma family of tumors (ESFT) contains a characteristic translocation the chimeric transcript of which is translated to become the EWS−FLI1 fusion protein. EWS−FLI1 regulates transcription and posttranscriptional splicing. Elimination of EWS−FLI1 protein from ESFT cells induces apoptosis and reduces xenograft tumor growth. Therefore the production of a biologically active recombinant EWS−FLI1 could lead to discoveries that would enhance our mechanistic understanding of ESFT. We have cloned, expressed, and purified a biologically active recombinant EWS−FLI1 in Escherichia coli using affinity column chromatography. A refolding procedure was required to render the recombinant EWS−FLI1 soluble in relatively native conditions. The structural alterations induced by the refolding procedure were monitored by SDS-gel electrophoresis, circular dichroism, and steady-state fluorescence spectroscopy. Recombinant EWS−FLI1 under native conditions approaches a largely unfolded conformation. Recombinant EWS−FLI1 protein under native conditions specifically binds to DNA and transcribes RNA. Our biologically active recombinant EWS−FLI1 oncoprotein will be useful to identify functional molecular partners and inhibitors.