Recombinant EWS-FLI1 oncoprotein activates transcription.
Recombinant EWS-FLI1 oncoprotein activates transcription.
复制标题
重组 EWS-FLI1 癌蛋白激活转录。
DOI：
10.1021/bi048776q
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Toretsky,JeffreyA
中科院分区：
文献类型：
--
作者：
Uren,Aykut;Tcherkasskaya,Olga;Toretsky,JeffreyA
尤文氏肉瘤肿瘤家族 (ESFT) 包含一种特征性易位，其嵌合转录物被翻译为 EWS-FLI1 融合蛋白。 EWS−FLI1 调节转录和转录后剪接。从 ESFT 细胞中消除 EWS−FLI1 蛋白会诱导细胞凋亡并减少异种移植肿瘤的生长。因此，生产具有生物活性的重组 EWS−FLI1 可能会带来一些发现，从而增强我们对 ESFT 机制的理解。我们在大肠杆菌亲和柱层析中克隆、表达和纯化了具有生物活性的重组EWS-FLI1。需要重折叠程序才能使重组 EWS-FLI1 在相对天然的条件下可溶。通过 SDS 凝胶电泳、圆二色性和稳态荧光光谱监测重折叠过程引起的结构变化。天然条件下的重组 EWS-FLI1 接近基本未折叠的构象。重组 EWS−FLI1 蛋白在天然条件下特异性结合 DNA 并转录 RNA。我们的生物活性重组 EWS−FLI1 癌蛋白将有助于识别功能性分子伴侣和抑制剂。
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 inEscherichia coliusing 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.