Attarpour Yazdi Mohammad Mehdi, Tofighi Nikky, Rajaee Taraneh, Ghahremanlou Marzieh, Adeli Ahmad, Azam Bolhassani, Azizi Mohammad, Davoudi Noushin
Department of Microbiology, Faculty of Medicine, Shahed University, Tehran, Iran.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Iran Biomed J. 2019 Jul;23(4):272-9. doi: 10.29252/.23.4.272. Epub 2018 Sep 17.
The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low-level eukaryote could represent a competitive alternative to the mammalian cell lines.
The full length of coding sequence of modified tPA TNKase (tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells.
The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase.
Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method.
生物治疗蛋白在哺乳动物细胞（如中国仓鼠卵巢细胞）中的表达在翻译后修饰方面具有高度同质性。尽管中国仓鼠卵巢细胞仍是市场上畅销生物治疗蛋白最常用的细胞系，但仍存在一些缺点，如培养基昂贵、生产周期长和药物成本高。最近，对一种新型利什曼原虫系统的研究证实，这种低等真核生物可能是哺乳动物细胞系的一种有竞争力的替代方案。
合成修饰型组织型纤溶酶原激活剂替奈普酶（TNKase）的全长编码序列，并将其克隆到墨西哥利什曼原虫T7-TR细胞的诱导表达载体中。
构建体的表达由四环素诱导型启动子驱动。表达盒中还添加了利什曼原虫分泌信号序列，以促进重组蛋白释放到培养基中。通过SDS-PAGE和蛋白质免疫印迹分析对分泌的重组蛋白进行了分析和确认。在该新型墨西哥利什曼原虫系统中，诱导后TNKase的表达水平为810 IU/mL，这意味着与墨西哥利什曼原虫先前的模型相比，表达百分比增加了两倍。TNKase活性与阿替普酶相当。
我们的结果表明，就其对纤维蛋白溶解的作用而言，表达的TNK（修饰型组织型纤溶酶原激活剂）在功能上与阿替普酶兼容。鉴于哺乳动物和墨西哥利什曼原虫在翻译后修饰方面的相似性，推测该系统能够像哺乳动物系统一样产生复杂蛋白质，如组织型纤溶酶原激活剂，且操作更简便、方法成本更低。
