Recombinant EWS-FLI1 oncoprotein activates transcription

Biochemistry. 2004 Oct 26;43(42):13579-89. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Circular Dichroism
  • Cloning, Molecular
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • 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
  • RNA-Binding Protein EWS / chemistry
  • RNA-Binding Protein EWS / genetics
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / physiology*
  • 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*


  • DNA-Binding Proteins
  • EWS-FLI fusion protein
  • FLI1 protein, human
  • Oncogene Proteins, Fusion
  • Proto-Oncogene Protein c-fli-1
  • RNA-Binding Protein EWS
  • Recombinant Fusion Proteins
  • Trans-Activators
  • Transcription Factors