Expression And Biological Characterization Of SAC-TRAIL Fusion Protein

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand(TRAIL), which is atypical member of the tumor necrosis factor(TNF)[1], shows selective pro-apoptosisactivity towards tumor cells and becomes an important targeted protein for therapy[2].However, in a variety of human cancers, high activity of the NF-κB regulator could resistthe pro-apoptosis effect of TRAIL[4].Prostate apoptosis response-4(Par-4), as a pro-apoptotic, and a tumor suppressorprotein[5], which exists both intracellular and extracellular of many kinds of cells showssignificant apoptosis-inducing activity selectively in tumor cells[6]. The critical domainSAC is both unique and indispensible for the pro-apoptotic activity of Par-4[7,8].In this study, we synthesized a codon-optimized SAC sequence chemically in terms ofthe codon usage bias in Escherichia coli and subcloned it into pMal-C2to increase theexpression. We connected SAC and TRAIL via (GGGGS)3in a head-to-tail mode,obtaining a single polypeptide chain. The recombinant SAC-TRAIL fusion protein waspurifed by Maltose Affinity Chromatography. Bioactivity analysis showed that the purifedSAC-TRAIL fusion protein displayed high level activity on apoptosis in NCL-H460-cells,SGC-7901-cells, BGC-823-cells, HeLa-cells, and it showed no toxicity to normalHUVEC-cells. Compared with TRAIL, recombinant SAC-TRAIL fusion protein stronglyreinforced the pro-apoptosis effect of TRAIL and resulted in enhancing the sensitization oftumor cells. Also, we found SAC-TRAIL fusion protein showed high bioactivity whenusing Escherichia coli as an expression host. This work provides a foundation for thedevelopment of SAC-TRAIL with clinical application prospects.Part one: Construction of Expression Vector, Expression and Purification ofRecombinant SAC and SAC-TRAIL Fusion Protein.The gene sequence of SAC and TRAIL was designed and cloned into the pMAL-c2 expression vector which had an efficient expression in the E.coli BL21under IPTGinduction. MBP-SAC-TRAIL was purified by amylose resin affinity purification columnand showed at the expected calculated molecular masses by SDS-PAGE and Immunoblotanalysis.Part two: Cell Death Assays and Acute Toxicity Tests.Three kinds of cells were treated by MBP-SAC-TRAIL. After over night incubation,cell death assays were assessed by the flow cytometry. Results implied thatMBP-SAC-TRAIL did not distinctly influence growth of normal cells (HUVEC) and hadsignificant effects of killing cancer cells (NCI-H460) superior to that of MBP-TRAIL.Results.The code of SAC-TRAIL gene in pMAL-SAC-TRAIL was confirmed to be correct.We obtained high expression levels of MBP-SAC-TRAIL and purified it with amyloseresin affinity purification column. MBP-SAC-TRAIL had significant biological activity ofinhibition of tumor growth but little of normal cells. Results showed thatMBP-SAC-TRAIL would has a better application prospect in clinical treatment of tumor.