Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum

Human Fas ligancl (hFasL) is a type II membrane protein that induces apoptosis in the Fas-bearing cells. Its special biological activity has the potential for the therapeutic use as an anti-cancer agent directed at enhancing apoptosis in tumor cells. In this study Escherichia coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amounts. An expression vector for hFasL production in E. coli was constructed based on plasmid pET32a(+). By cultivation of the hFasL-producing E. coli clone on LB medium and induction with IPTG, a hFasL concentration of 1.0 mg L-1 was achieved. D. discoideum strain AX3-hFasL-H was cultured in a conventional stirred bioreactor on an improved synthetic medium using a simple fed-batch strategy, and cell densities of up to 8.3 x 10(7) cells/mL and a maximum hFasL concentration of 420 mu g/L were obtained. 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. They showed similar biological activities in inducing apoptosis in Fas-expressing cells. (C) 2012 Elsevier B.V. All rights reserved.Program for New Century Excellent Talents in Fujian Province, PR China; National Natural Science Foundation of China [31071488, 20736008, 20928006