Production of the cell permeable T34A/C84A dominant-negative survivin protein a core unit of the novel survivin-loaded nanoparticles for human cancer treatment

  • 張 詠傑

Student thesis: Master's Thesis

Abstract

Survivin is a member of the inhibitor-of-apoptosis (IAP) family Unlike other family members survivin is primarily expressed in fetal and cancerous tissue rather than in differentiated adult tissue Overexpression of survivin is correlated with poor prognosis and drug resistance in cancer patients Although survivin has been proposed as a promising molecular target for cancer treatment it is widely believed that survivin is only a semi-druggable target In fact only a few survivin inhibitors have been developed and only one survivin specific small molecular inhibitor YM155 has successfully reached phase II clinical trials in the past decade Unfortunately recently studies demonstrated that YM155 is a substrate of the multiple-drug resistant protein (MDR-1) and this property actually limits its clinical potential Therefore it is quite urgent to develop a new survivin specific inhibitor The aim of this study is to evaluate the molecular mechanism of anti-cancer effects of various dominant-negative survivin mutants and to develop a novel oral and clinical applicable survivin specific macromolecular inhibitor a cell permeable dominant-negative survivin protein loaded nanoparticles for the treatment of cancer MTT cell viability and Western blot analysis revealed that the T34A/C84A (double point mutation) dominant-negative survivin exhibits higher anti-cancer effects as compared to both T34A and C84A (single point mutation) dominant-negative survivin in vitro A cell-permeable dominant-negative form of survivin (GST-tagged dNSurR9-T34A/C84A) comprising a N-terminal GST-tag and nine C-terminal arginine residues fused to the T34A/C84A dominant-negative survivin mutant was constructed The GST-tagged recombinant survivin protein was expressed using bacteria [E coli BL21(DE3) codon plus RIPL strain] protein expression system The expressed insoluble GST-tagged dNSurR9-T34A/C84A proteins were solubilized by varying the induction temperatures IPTG concentrations expression durations and lysis buffer compositions The solubilized recombinant proteins were purified using the GST affinity chromatography and cation-exchange chromatography Results of the sodium dodecyl sulfate polyacrylamide gel electrophoresis and the Western blot analysis showed that the recombinant proteins was successfully solubilized and purified under the optimized conditions Further studies are needed to determine the anti-cancer activity of the purified protein before nanoparticle encapsulations
Date of Award2014 Jul 29
Original languageEnglish
SupervisorChun Hei Antonio Cheung (Supervisor)

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