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- Title
Optimization Studies on Prokaryotic Cell Expression of the Human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL).
- Authors
Hao, Lin; Zhang, Zhi-Guo; Shi, Zheng-Duo; Pang, Kun; Zhang, Jun-Jie; Dong, Yang; Han, Cong-Hui
- 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/TRAIL 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-rTRAIL 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.
- Subjects
TUMOR necrosis factors; APOPTOSIS; DENATURATION of proteins; ESCHERICHIA coli; SODIUM dodecyl sulfate; POLYACRYLAMIDE gel electrophoresis; AFFINITY chromatography
- Publication
Cell Biochemistry & Biophysics, 2015, Vol 73, Issue 2, p275
- ISSN
1085-9195
- Publication type
Article
- DOI
10.1007/s12013-015-0596-6