Optimization Studies on Prokaryotic Cell Expression of the Human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL).

Abstract

The aim of the study was to optimize the in vitro induction and expression of the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and also study the processes of its denaturation, renaturation, and purification. The pGEX-6P-1/TRAIL114-281 plasmid was induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli BL21 (DE3), and the expressed target protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein expressed in the form of inclusion body was first denaturalized and then renaturalized by dilution and dialysis technique. GST-rTRAIL114-281 fusion protein was purified by Glutathione-Superflow Resin affinity chromatography and confirmed by Western blot. The molecular weight of GST-rTRAIL expressed in E. coli BL21 (DE3) was approximately 40kDa. GST-rTRAIL was mainly expressed in the form of inclusion bodies. An optimum expression was induced by IPTG at a concentration of 0.2mM for 8h at 37°C. Glutathione-Superflow Resin affinity chromatography yielded the purified GST-rTRAIL protein which was confirmed by Western blot using anti-GST mouse monoclonal antibody. The optimum prokaryotic cell expression of the human GST-rTRAIL was obtained by 0.2mM IPTG induction for 8h at 37°C. The denatured inclusion body protein can be refolded by dilution and dialysis and purified by Glutathione-Superflow Resin affinity chromatography.