通过毕赤酵母表达系统获得高纯度的重组人血管内皮生长因子 (rhVEGF165)

Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris

摘要:

血管内皮生长因子 (Vascular endothelial growth factor，VEGF165) 是一种高度特异性的促血管内皮细胞生长因子，高纯度的VEGF165对于抗肿瘤药物和生物标志物研发检测试剂必不可少。目前关于VEGF165的异源表达方法，纯化步骤多且产物纯度不高。以毕赤酵母表达系统为基础，构建人血管内皮生长因子 (VEGF165) 多拷贝的表达载体。按照酵母密码子偏好性优化人血管内皮生长因子基因 (vegf165) 的密码子，在毕赤酵母BBPB表达载体基础上，用Biobrick生物积块的方法，构建以Pgap为启动子的五拷贝rhVEGF165表达载体，同时添加组氨酸标签。利用His标签和VEGF165自身的肝素结合结构域，仅用两步亲和层析纯化得到纯度高于98%的rhVEGF165蛋白。rhVEGF165纯化后浓度为0.45 mg/mL，且具有生物学活性。该异源表达策略简化了rhVEGF165的纯化步骤，rhVEGF165具有天然VEGF165的生物学活性，且纯度达到目前文献报道的最高水平。

Abstract:

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.