[Cloning, expression and purification of human TNF-related apoptosis-inducing ligand in Escherichia coli].
OBJECTIVE
To clone human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, 114-281) cDNA and develop an inducible system for expression in E. coli.


METHODS
The human TRAIL (114-281) cDNA was amplified with the total RNA from the human peripheral blood monocytes by RT-PCR. The cDNA was inserted into pMD T vector. The selected integrants were confirmed by PCR screen, digestion with restriction enzymes and DNA sequencing. Then, the insert of human TRAIL cDNA was subcloned into prokaryotic expression vector pET28a. The construction of prokaryotic expression vector was proofed correct by RT-PCR and digestive identification. The recombinant protein was induced by IPTG (isopropyl-beta-D-thiogalactopyranoside) . The sTRAIL inclusion bodies were refolded by dilution method. Refolded protein was purified with column chromatography.


RESULTS
Agarose gel electrophoresis of product of RT-PCR revealed a band around 500bp, which was expected. The positive integrants were confirmed by PCR screen and digestion with restriction enzymes. The same band as RT-PCR product was showed by PCR screen and digestion with restriction enzymes. Then the selected integrants were confirmed by DNA sequencing. The sequence was checked in GenBank. The construction of prokaryotic expression vector pET 28a was proofed correct by RT-PCR and digestive identification. TRAIL protein was successfully induced by IPTG in E. coli BL21. The results also showed that the protein was expressed as inclusion bodies. After the sTRAIL inclusion bodies were solubilized and refolded, the protein expressed was purified with one band, which was about 20kD, analyzed by SDS-PAGE.


CONCLUSIONS
These results suggested that the hsTRAIL was cloned, expressed and purified in the present study. Significant quantities of TRAIL produced by this method should allow further studies in determining the physiological significance and function of TRAIL in the future.
Yao
Gen-hong
GH
Department of Transfusion, Jinling Hospital, Nanjing 210002, China.
Luan
Jian-feng
JF
Ye
Dong
D
Lei
Qian-hong
QH
Zhu
Pei-yuan
PY
Jin
Jie
J
Hou
Ya-yi
YY
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand