Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in <Emphasis Type="Italic">E. coli</Emphasis> with Inhibiting Effect on Endothelial Cells |
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Authors: | Qing Gong Xia Yang Weibin Cai Guoquan Gao Zhonghan Yang |
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Institution: | (1) Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2 Road, 510080 Guangzhou, Guangdong, China; |
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Abstract: | PEDF34, a functional epitope of pigment epithelium-derived factor (PEDF), obtained by chemical synthesis previously, shows
potential anti-angiogenesis activity described before. We perform a novel method in this study for the expression and purification
of recombinant PEDF34 in E. coli, and make it convenient, soluble and high yield to obtain this small peptide of PEDF. Human PEDF34 gene was cloned into the
fusion-protein expression vector pGEX-4T-1, and the recombinant plasmid was transformed into E. coli strain BL21-DE3. GST-PEDF34 fusion protein was expressed, purified using chromatograph and identified by Western blotting.
The purified fusion protein was digested by thrombin, and the small PEDF34 peptide was isolated by ultrafiltration. Circular
dichroism (CD) analysis identified that secondary structure of PEDF34 mainly characterizes as α-helix. The 34-AA small peptide
could cell-type-specifically inhibit viability of HUVECs in a dose-dependent manner and induce apoptosis of HUVECs. These
results suggested that this type of recombinant PEDF34 may have potential in the treatment of angiogenesis-related diseases
such as solid tumor. |
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