Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in E. coli with Inhibiting Effect on Endothelial Cells
Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in E. coli with Inhibiting Effect on Endothelial Cells
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具有内皮细胞抑制作用的色素上皮衍生因子功能表位在大肠杆菌中的表达和纯化
DOI：
10.1007/s10930-010-9236-6
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发表时间：
2010-04-01
期刊：
影响因子：
3
通讯作者：
Yang, Zhonghan
中科院分区：
文献类型：
--
作者：
Gong, Qing;Yang, Xia;Yang, Zhonghan
PEDF34是色素上皮衍生因子（PEDF）的功能表位，先前通过化学合成获得，具有潜在的抗血管生成活性。本研究提出了一种在大肠杆菌中表达和纯化重组PEDF34的新方法，使其方便、易溶、产率高。将人PEDF34基因克隆到融合蛋白表达载体pGEX-4T-1中，并将重组质粒转化到大肠杆菌BL21-DE3中。表达GST-PEDF34融合蛋白，经色谱纯化，Western blotting鉴定。纯化后的融合蛋白经凝血酶消化，超滤分离pef34小肽。圆二色性（CD）分析表明，PEDF34的二级结构主要为α-螺旋结构。34-AA小肽能以剂量依赖性的方式特异性抑制HUVECs的活力，诱导HUVECs凋亡。这些结果表明，这种类型的重组PEDF34可能在治疗血管生成相关疾病如实体瘤方面具有潜力。
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.