Cloning, expression, and purification of the recombinant pro-apoptotic dominant-negative survivin T34A-C84A protein in Escherichia coli

Shing Ling Tsai, Yung Chieh Chang, Sailu Sarvagalla, Shuying Wang, Mohane Selvaraj Coumar, Chun Hei Antonio Cheung

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Abstract

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.

Original languageEnglish
Pages (from-to)73-83
Number of pages11
JournalProtein Expression and Purification
Volume160
DOIs
Publication statusPublished - 2019 Aug

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All Science Journal Classification (ASJC) codes

  • Biotechnology

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