Wei Yu-ying, Song Chao-jun, Sun Yuan-jie, Gong Jiu-yu, Jin Bo-quan, Yang An-gang, Yang Kun
Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Sep;24(9):870-2.
To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein.
The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.coli expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E.coli BL21 (DE3) pLysS and induced to express fusion protein VEGFR2 D3.4/GST with IPTG. The expressed protein was purified by washing in urea and detected by SDS-PAGE and Western blot.
SDS-PAGE analysis showed that a novel protein with the expected molecular mass (M(r);) about 46 000 was expressed with the inducement of IPTG. And it existed mostly in the form of inclusion body. Grayscale scanning showed that the expressed VEGFR2 D3.4/GST fusion protein accounted for 38.6% of the total bacterium protein. After the purified product was washed by urea, its purity reached 87.1%. Western blot confirmed the recombinant protein was VEGFR2 D3.4/GST fusion protein.
High purification VEGFR2 D3.4/GST fusion protein is obtained through the E.coli expression system.
构建用于表达VEGFR2 D3.4/GST融合蛋白的原核表达载体，并表达和纯化该融合蛋白。
合成人VEGFR2基因片段第三和第四细胞外结构域的编码序列，并亚克隆至大肠杆菌表达载体pGEX4T-1中GST片段的下游，构建重组质粒pGEX4T-VEGFR D3.4。然后将该质粒转化至大肠杆菌BL21(DE3)pLysS中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达融合蛋白VEGFR2 D3.4/GST。通过尿素洗涤纯化表达的蛋白，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）进行检测。
SDS-PAGE分析显示，经IPTG诱导表达出一种预期分子量（M(r)）约为46 000的新蛋白，且主要以包涵体形式存在。灰度扫描显示，表达的VEGFR2 D3.4/GST融合蛋白占细菌总蛋白的38.6%。纯化产物经尿素洗涤后，纯度达到87.1%。Western blot证实该重组蛋白为VEGFR2 D3.4/GST融合蛋白。
通过大肠杆菌表达系统获得了高纯度的VEGFR2 D3.4/GST融合蛋白。
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