Production Of Soluble Human Fas Ligand By Escherichia Coli And Dictyostelium Discoideum

Human Fas ligand(hFasL) is a cell apoptosis factor belonging to the tumor necrosis factor(TNF) family.It is a typeâ…¡membrane protein that induces apoptosis in the Fas-bearing cells.According to its special biological activity,hFasL has the potential therapeutic use as an anti-cancer agent directing at enhancing apoptosis in tumor cells.In this study E.coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amount.An expression vector for hFasL production in E.coli was constructed based on plasmid pET32a(+),whose TrxA Tag improve the recombinant protein to be expressed in the soluble form.DNA encoding the extracellular region of human Fas ligand(amino acids 141-281) was PCR-amplified from plasmid pMB74-hFasL and cloned into pET32a(+).The recombinant plasmid pET32a-hFasL was then transformed into E.coli BL21(DE3) for protein expression and checked through DNA sequencing.The sequencing result showed that the recombinant plasmid pET32a-hFasL was successfully constructed.The clone E.coli BL21(DE3) harboring the pET32a-hFasL was cultured in shake flasks using LB medium to verify the protein expression.The inducing conditions were optimized including induction temperature,concentration of IPTG and inducing time,and under the best condition a hFasL concentration of 1.0 mg L^-1 was reached.D.discoideum stain AX3-FH was cultured in a conventional stirred bioreactor on synthetic SIH medium,and cell densities of up to 8.3×10⁷ mL^-1 and a maximum hFasL concentration of 420μg L^-1 were obtained after 80 hours of cultivation.Using Ni-NTA affinity chromatography purification,two kinds of recombinant hFasLs from E.coli and D.discoideum were purified with a purity of 94%and 90%,respectively.The cytotoxic activities of recombinant hFasLs were determined by MTT method.MTT reaction conditions including cell density,concentration of MTT reagent and MTT reaction time were optimized.Under the best MTT reaction condition,these two kinds of recombinant hFasLs showed similar biological activities in inducing apoptosis in Pulmonary Carcinoma(A549) and Cervical Carcinoma(Hela) cells.