Tsai Shing-Ling, Chang Yung-Chieh, Sarvagalla Sailu, Wang Shuying, Coumar Mohane Selvaraj, Cheung Chun Hei Antonio
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, 605014, India.
Protein Expr Purif. 2019 Aug;160:73-83. doi: 10.1016/j.pep.2019.04.003. Epub 2019 Apr 17.
Survivin is a well-known inhibitor-of-apoptosis proteins family member and a promising molecular target for anti-cancer treatment. However, it is widely accepted that survivin is only a "semi-druggable" target and development of survivin-specific small molecule inhibitors has shown to be difficult. In this study, we demonstrated that a histidine-tagged survivin T34A-C84A mutated protein (T34A-C84A-dNSur-His) can be produced using a bacterial recombinant protein expression system [E. coli ArcticExpress (DE3) cells] and solubilized using 1% (w/v) Sarkosyl. In addition, we showed that the purified T34A-C84A-dNSur-His protein formed dimers as predicted by in silico protein structure and molecular dynamics analysis. Importantly, results of the MTT assay revealed that the purified recombinant protein was biologically active in decreasing the viability of the human MDA-MB-231 breast adenocarcinoma and MIA-PaCa pancreatic carcinoma cells in vitro. Furthermore, the purified T34A-C84A-dNSur-His protein, but not of the histidine-peptide, induced apoptosis (i.e. caspase-9 activation and DNA fragmentation) in MDA-MB-231 cells at concentrations from 50 to 400 nM. In conclusion, our study provides a protocol of producing a biologically active survivin-targeting macromolecule, T34A-C84A-dNSur-His, which can be used as a tool for studying the molecular and cellular roles of survivin in cells. T34A-C84A-dNSur-His is also a potential therapeutic agent for augmenting cancer therapy.
生存素是一种著名的凋亡抑制蛋白家族成员，也是抗癌治疗中一个很有前景的分子靶点。然而，人们普遍认为生存素只是一个“半可成药”靶点，开发针对生存素的小分子抑制剂已证明具有难度。在本研究中，我们证明了一种带有组氨酸标签的生存素T34A - C84A突变蛋白（T34A - C84A - dNSur - His）可以使用细菌重组蛋白表达系统[大肠杆菌ArcticExpress (DE3)细胞]来生产，并能用1%（w/v）十二烷基肌氨酸钠溶解。此外，我们表明纯化后的T34A - C84A - dNSur - His蛋白如计算机模拟蛋白质结构和分子动力学分析所预测的那样形成了二聚体。重要的是，MTT试验结果显示，纯化后的重组蛋白在体外可降低人MDA - MB - 231乳腺腺癌和MIA - PaCa胰腺癌细胞的活力，具有生物学活性。此外，纯化后的T34A - C84A - dNSur - His蛋白（而非组氨酸肽）在浓度为50至400 nM时可诱导MDA - MB - 231细胞凋亡（即半胱天冬酶 - 9激活和DNA片段化）。总之，我们的研究提供了一种生产具有生物学活性的靶向生存素大分子T34A - C84A - dNSur - His的方案，该大分子可作为研究生存素在细胞中的分子和细胞作用的工具。T34A - C84A - dNSur - His也是一种增强癌症治疗效果的潜在治疗剂。
