Optimization Studies on Prokaryotic Cell Expression of the Human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL).
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.
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.|Department of Urology, Clinical College, Xuzhou Medical College, Xuzhou, 221009, Jiangsu, China.
Lin
L
Hao
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.|Department of Urology, Clinical College, Xuzhou Medical College, Xuzhou, 221009, Jiangsu, China.
Zhi-Guo
ZG
Zhang
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.|Department of Urology, Clinical College, Xuzhou Medical College, Xuzhou, 221009, Jiangsu, China.
Zheng-Duo
ZD
Shi
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.
Kun
K
Pang
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.
Jun-Jie
JJ
Zhang
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China.
Yang
Y
Dong
Department of Urology, Xuzhou Center Hospital, Xuzhou, 221009, Jiangsu, China. hanchdoctor@qq.com.|Department of Urology, Clinical College, Xuzhou Medical College, Xuzhou, 221009, Jiangsu, China. hanchdoctor@qq.com.
Cong-Hui
CH
Han
D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand
Inclusion body; Prokaryotic expression; Purification; TRAIL