VEGFR2 D3.4/GST融合蛋白在大肠杆菌的表达、纯化及鉴定

Prokaryotic expression,purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli

目的:构建人血管内皮细胞生长因子II型受体(VEGFR2)胞膜外区第三及第四个免疫球蛋白样结构域与GST融合蛋白VEGFR2 D3.4/GST基因的原核表达载体,在大肠杆菌中表达并纯化该融合蛋白。方法:由公司合成VEG-FR2胞膜外区第三、第四免疫球蛋白样结构域氨基酸的编码序列,克隆入原核表达载体pGEX-4T1中GST标签的下游,构建重组表达载体pGEX4T-VEGFR2 D3.4,将该载体转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达VEGFR2 D3.4/GST融合蛋白,经不同浓度尿素洗脱纯化后,表达产物用SDS-PAGE和Western blot进行鉴定。结果:SDS-PAGE分析表明,表达产物的相对分子质量(Mr)为46 000,与理论值相符,主要以包含体形式存在;灰度扫描分析融合蛋白的表达量占菌体蛋白总量的38.6%,纯化产物纯度最高为87.1%,Western blot证实该蛋白为VEGFR2 D3.4/GST融合蛋白。结论:利用大肠杆菌表达系统,获得了较高纯度的包含体形式VEGFR2 D3.4/GST融合蛋白。

AIM： To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein. METHODS： 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. coil expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E. coil 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. RESULTS： SDS-PAGE analysis showed that a novel protein with the expected molecular mass （ Mt ） 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. CONCLUSION： High purification VEGFR2 D3.4/GST fusion protein is obtained through the E. coli expression system.