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Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in E. coli with Inhibiting Effect on Endothelial Cells

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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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Abbreviations

PEDF:

Pigment epithelium-derived factor

HUVECs:

Human umbilical vein endothelial cells

LR:

Laminin receptor

PNPLA2:

Phospholipase A2/nutrin/patatin-like phospholipase domain-containing 2

GST:

Glutathione-S-transferase

PI:

Propidium iodide

E. coli :

Escherchia coli

CD:

Circular dichroism

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Acknowledgments

This study was supported by National Nature Science Foundation of China, Grant Number: 30570372, 30600724, 30700120, 30872980, 30971208, 30973449; Key Sci-tech Research Project of Guangdong Province, China, Grant Number: 2008B080703027; Key Sci-tech Research Project of Guangzhou Municipality, China, Grant Number: 2007Z3-E5041, 2008Z1-E231; National Key Sci-Tech Special Project of China, Grant Number: 2009ZX09103-642.

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Authors and Affiliations

  1. Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2 Road, 510080, Guangzhou, Guangdong, China

    Qing Gong, Xia Yang, Weibin Cai, Guoquan Gao & Zhonghan Yang

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  1. Qing Gong
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  2. Xia Yang
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  3. Weibin Cai
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  4. Guoquan Gao
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  5. Zhonghan Yang
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Correspondence to Zhonghan Yang.

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Gong, Q., Yang, X., Cai, W. et al. Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in E. coli with Inhibiting Effect on Endothelial Cells. Protein J 29, 167–173 (2010). https://doi.org/10.1007/s10930-010-9236-6

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