Tavallaei Omid, Bandehpour Mojgan, Nafissi-Varcheh Nastaran, Kazemi Bahram
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. ; Biotechnology Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2015 Spring;14(2):617-26.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）是肿瘤坏死因子家族的成员，是一种有趣的配体，它能选择性地诱导肿瘤细胞凋亡，因此已被开发用于癌症治疗。这种配体已由多种宿主如大肠杆菌产生。然而，在大肠杆菌细胞质中进行蛋白质表达会导致诸如错误折叠、生物活性降低、包涵体形成以及复杂的下游处理等问题。本研究的目的是开发一种表达系统，用于生产分泌到大肠杆菌周质而非细胞质中的重组TRAIL。通过使用重叠延伸PCR，将OmpA信号序列与TRAIL cDNA融合，然后将OmpA - TRAIL片段克隆到pET - 22b质粒中。通过PCR和DNA测序对该构建体进行了确认。在大肠杆菌BL21（DE3）中诱导启动子，并使用渗透压休克法释放周质表达的蛋白质。SDS - PAGE分析表明，约37%的重组TRAIL转移到了周质中，并且通过蛋白质印迹分析确认了其身份。最后，使用MTT法证实了TRAIL对HeLa细胞系的细胞毒性活性。结果表明，我们的表达系统可能有助于在周质空间中生产TRAIL。
