Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran.
Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran.
Int J Biol Macromol. 2019 Oct 15;139:1028-1034. doi: 10.1016/j.ijbiomac.2019.08.080. Epub 2019 Aug 9.
We report cloning and expressing of recombinant human VEGF-A, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A production. Supplementing the Pichia pastoris cell culture medium with Mg, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A production was with the addition of PBA, PS 20, and MgSO. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.
我们报告了重组人 VEGF-A 的克隆和表达，该蛋白在 N 端与来自里氏木霉的 Hydrophobin II（HFBII）融合，在毕赤酵母中表达。我们使用 SDS-PAGE 和针对 VEGF-A 的 ELISA 验证了构建体，并根据 HFBII 对应物有效地进行了蛋白质纯化和浓缩，并使用非离子表面活性剂 X-114 进行了双水相系统（ATPS）。我们研究了各种培养基添加剂的影响以及正因子的相互作用，以增加重组 HFBII-VEGF-A 的产量。在毕赤酵母细胞培养基中添加 Mg、聚山梨酯 20（PS 20）和 4-苯基丁酸（PBA）可提高嵌合蛋白的表达。正交实验表明，达到最大 HFBII-VEGF-A 产量的最佳条件是添加 PBA、PS 20 和 MgSO。在此条件下，目标蛋白的产量比无添加剂培养基中的产量高 4.5 倍。总的来说，我们生产嵌合 HFBII-VEGF-A 并在 ATPS 中选择性捕获它的方法有望实现无需繁琐下游处理的大规模蛋白质生产。
