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.