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

• DOI: 10.1007/s12013-015-0596-6
• Publication Date: 2015-03-03T18:23:49Z
• Authors: Lin Hao; Zhi-Guo Zhang; Zheng-Duo Shi; Kun Pang; Jun-Jie Zhang; Yang Dong; Cong-Hui Han
• Keywords: Inclusion Bodies; 0301 basic medicine; Protein Denaturation; Recombinant Fusion Proteins; Blotting, Western; Chromatography, Affinity; Protein Refolding; 3. Good health; TNF-Related Apoptosis-Inducing Ligand; 03 medical and health sciences; Escherichia coli; Humans; Electrophoresis, Polyacrylamide Gel; Cloning, Molecular; Plasmids

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 40 kDa. GST-rTRAIL was mainly expressed in the form of inclusion bodies. An optimum expression was induced by IPTG at a concentration of 0.2 mM for 8 h 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 0.2 mM IPTG induction for 8 h at 37 °C. The denatured inclusion body protein can be refolded by dilution and dialysis and purified by Glutathione-Superflow Resin affinity chromatography.