Expression and purification of functional epitope of pigment epithelium-derived factor in E. coli with inhibiting effect on endothelial cells.
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 alpha-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.
Gong
Qing
Q
Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2 Road, 510080, Guangzhou, Guangdong, China.
Yang
Xia
X
Cai
Weibin
W
Gao
Guoquan
G
Yang
Zhonghan
Z
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D014462:Ultrafiltration; D014471:Umbilical Veins / Q000166:cytology; D000099174:Pigment Epithelium-Derived Factor
D000939:Epitopes; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor