Expression, refolding, and purification of a truncated human Delta-like1, a ligand of Notch receptors
Expression, refolding, and purification of a truncated human Delta-like1, a ligand of Notch receptors
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截短的人 delta-like1（Notch 受体配体）的表达、重折叠和纯化
DOI：
10.1016/j.pep.2008.02.004
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发表时间：
2008-06-01
影响因子：
1.6
通讯作者：
Geng, Xin-Du
中科院分区：
文献类型：
--
作者：
Shi, Zhan-Xia;He, Fei;Geng, Xin-Du
Notch信号通路在胚胎和出生后发育中的增殖、凋亡和细胞命运特化中起关键作用，并且是人类疾病如癌症的潜在治疗靶点。为了在大肠杆菌中表达和纯化人δ-like 1（hDll 1）的可溶性片段，我们克隆了hDll 1的两个胞外片段[hDll 1（127-225）和hDll 1（26-225）]。hD 111（127-225）片段在大肠杆菌中成功表达为GST融合蛋白（GST-hD 111）。表达产物经IPTG诱导后以包涵体形式表达，经亲和层析和分子排阻层析复性纯化。纯化的GST-hDll 1纯度大于95%，分子量为39 kDa。报告基因分析表明GST-hDll 1可激活一个依赖于Notch激活的报告基因。因此，利用大肠杆菌表达系统和不同的层析系统，我们成功地表达、复性和纯化了具有生物活性的GST-hDll 1，这可能对Notch通路的治疗和研究有潜在的用途。(C)2008年爱思唯尔公司All rights reserved.
The Notch signaling pathway plays a pivotal role in proliferation, apoptosis, and cell fate specification in both embryonic and postnatal development, and is a potential therapeutic target for human diseases such as cancer. To express in Escherichia coli and purify soluble fragment of human Delta-like1 (hDl11), we cloned two extracellular fragments of hDI11 [hDll1 (127-225) and hDll1 (26-225)]. The hDl11 (127-225) fragment was successfully expressed in E coli as a GST fusion protein (GST-hDll1). The GST-hDll11 protein, which was expressed as inclusion bodies after induction by IPTG, was refolded and purified simultaneously using affinity chromatography and size exclusion chromatography. The purified GST-hDll1 was of more than 95% purity, and had a molecular weight of 39 kDa. Reporter assay showed that GST-hDll1 could activate a reporter gene that is dependent on Notch activation. Therefore, using the E coli expression system and different chromatography systems, we successfully expressed, refolded, and purified a biologically active GST-hDll1, which might be potentially useful for therapy and studying the Notch pathway. (C) 2008 Elsevier Inc. All rights reserved.