Studies On Prokaryotic Expression System Based On Procaspase-8 DEDs And Cell Lines For Screening Anti-cancer Drugs

TRAIL(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)is a member of the Tumor necrosis factor family.As its ability of selectively inducing apoptosis of malignant and virus-infected cells,without significant side effects on normal cells,TRAIL has been attracting a rising attention from scholars in recent years.Moreover,TRAIL has been identified as a potential target for drug development to treat Alzheimer's disease,atherosclerosis and so on.Therefore,according to the interaction between those two crucial proteins Procaspase-8 DEDs/FADD in the TRAIL apoptotic pathway,we hope to obtain the key amino acid sites of the interaction of the Procaspase-8 DEDs protein with FADD,to warrant the future potential antagonist as the therapeutic agent against the Alzheimer's disease treatment.In addition,on the basis of Procaspase-8 DEDs,cell lines contain the fluorescence protein(GFP)fusion protein obtained by constructing a stable expression of this fusion protein can be used to screen natural products with anticancer activities.Our preliminary studies revealed the wild type Procaspase-8 DEDs protein in eukaryotic cells and prokaryotic cells are not conducive for subsequent experiments.After reviewing on abundant relevant studies and together with our studies,it has been shown that the aggregation between each wild-type Procobase-8 DEDs could result in death effect filament(DEFs).And the fluorescent aggregation point spontaneously formed could in turn promotes apoptosis in eukaryotic cells.In the prokaryotic system,its undesired aggregating tendency and low solubility also present a big challenge for subsequent experiments.In this study,three sites F122,L123 and 1128 of the wild-type(WT)Procaspase-8 DEDs protein were subjected to simultaneous site-directed mutagenesis,and the mutant gene fragments were constructed into expression vectors pET-28a(+)and pCMV6-AC-GFP;Transforming prokaryotic plasmid into E.coli Transetta(DE3),we have successfully acquired the prokaryotic expression strain.The soluble expression of Procaspase-8 DEDsF122G/L123G/I128D protein was significantly higher than that of wild-type Procaspase-8 DEDs by induced expression with IPTG;The recombinant eukaryotic plasmid was transfected into HCT116 cells.Comparing with the expression of Procaspase-8 DEDs protein,mutant protein does not tend to form death effector filaments(DEFs).Later the drug screening experiments of HepG2 cell lines transfected with procopase-8 DEDsF122G/L123G/I128D eukaryotic plasmids were carried out and found to show a significant green fluorescence aggregation point with time.However,it was later found that the green fluorescence aggregation points were occurred in HepG2 cells transfected procaspase-8 DEDsF122GLI23GI128D eukaryotic plasmids without TRAIL stimulation,but ultimately the cell line died.So the soluble expression of Procaspase-8 DEDs F122G/L123G/I128D protein in E.coli could be enhanced by site-directed mutagenesis,and the soluble target protein could be assembled by Ni2+ protein purification column and protein concentration column.Meanwhile,in eukaryotic cells,although Procaspase-8 DEDsF122G/L123G/I128D protein decrease the occurance of death effector filaments in a way,cell lines transfected with procaspase-8 DEDsF122GLI23GI128D eukaryotic fluorescent plasmids worth further investigated for drug screening.This study has laid a foundation for the further study of Procaspase-8 DEDs protein.