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

Recombinant EWS-FLI1 oncoprotein activates transcription.

Author information

1
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

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.

PMID:
15491164
DOI:
10.1021/bi048776q
[Indexed for MEDLINE]

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