title: Recombinant EWS-FLI1 oncoprotein activates transcription.
abstract: 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.
authors.affiliation: Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Research Building, Room W316, 3970 Reservoir Road, N.W., Box 571469, Washington, DC 20057-1469, USA. au26@georgetown.edu
authors.forename: Aykut
authors.initials: A
authors.lastname: Uren
authors.forename: Olga
authors.initials: O
authors.lastname: Tcherkasskaya
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Toretsky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D012512:Sarcoma, Ewing / Q000737:chemistry / Q000188:drug therapy / Q000235:genetics; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C106031:EWS-FLI fusion protein; C078433:FLI1 protein, human; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors