Department of Laboratory Sciences, Faculty of Para-Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Biotechnol Prog. 2019 Jul;35(4):e2819. doi: 10.1002/btpr.2819. Epub 2019 Apr 25.
Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut strain and KM71H as a Mut strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3-4 days. The highest expression was obtained at the range of 2-3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25-37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7-9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure.
葡萄球菌激酶 (SAK) 是一种很有前途的溶栓药物，可用于治疗患有血栓形成障碍的患者。为了提高 SAK 的效力并最大程度地减少血管再闭塞，一种新的构建体通过 20 个氨基酸接头融合了 SAK 基序和采采蝇凝血酶抑制剂 (TTI)，接头带有 2 个 RGD (2×精氨酸-甘氨酸-天冬氨酸通过与血小板整合素受体结合抑制血小板聚集)，经密码子优化并在毕赤酵母 GS115 中作为 Mut 株和 KM71H 作为 Mut 株进行表达。融合蛋白的最佳表达条件和纤维蛋白溶解活性进行了优化，并与 rSAK 进行了比较。在 2.5%甲醇刺激 72 小时后，设计的构建体的表达水平达到了培养基的 175mg/L，并且在 3-4 天内保持稳定。在 2-3%甲醇的范围内获得了最高的表达。SAK-2RGD-TT(相对活性>82%)在 25-37°C 时比 rSAK(相对活性为 93%)更活跃。此外，它在 pH7-9 的范围内显示出相对活性>80%。Western blot 分析显示，两个条带的大小分别约为 27 和 24kDa，比例为 5:3。未经纯化和纯化蛋白的 SAK-2RGD-TTI 的比纤溶活性分别为 8269U/mg 和 19616U/mg。在培养基中使用衣霉素进行糖基化处理可使 SAK-2RGD-TTI 的纤溶活性提高 2.2 倍。因此，与 rSAK 相比，在相同等摩尔比例下，添加 RGD 和 TTI 片段可以提高纤溶活性。此外，毕赤酵母可以被认为是表达具有高活性的可溶性 SAK-2RGD-TTI 的有效宿主，而不需要复杂的纯化程序。
