Expression and purification of soluble recombinant Human Endostatin in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Cuihong Du Xiaoping Yi Yuanxing Zhang |
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Institution: | (1) Departments of Medicine and Cancer Biology, and Center for Matrix Biology, Vanderbilt University, Nashville, TN, 37232, USA |
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Abstract: | Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and
angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin)
in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production
of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin
fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant
fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and
NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography.
As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity
of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach
to produce soluble and biologically active rhEndostatin. |
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