Expression, refolding, and purification of a truncated human Delta-like1, a ligand of Notch receptors.
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 (hDll1), we cloned two extracellular fragments of hDll1 [hDll1 (127-225) and hDll1 (26-225)]. The hDll1 (127-225) fragment was successfully expressed in E. coli as a GST fusion protein (GST-hDll1). The GST-hDll1 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 39kDa. 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.
Shi
Zhan-Xia
ZX
Institute of Modern Separation Sciences, Department of Chemistry, Northwest University, Provincial Key Laboratory of Shaanxi, 229 Tai Bai North Road, Xian 710069, China.
He
Fei
F
Wang
Li-Li
LL
Liang
Ying-Min
YM
Han
Hua
H
Wang
Chao-Zhan
CZ
Zhao
Qun
Q
Geng
Xin-Du
XD
D002135:Calcium-Binding Proteins; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D017510:Protein Folding; D051880:Receptors, Notch / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D002135:Calcium-Binding Proteins; C512941:DLK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase