[Gene expression and primary functional characterization of human DR5 extracellular fragment].
OBJECTIVE
To acquire human DR5 extracellular fragment with bioactivity.


METHODS
Total RNA was prepared from Jurkat cells by Trizol. Human DR5 extracellular fragment gene was amplified by RT-PCR, cloned into pGEM-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pET30a with a His-tag at the amino terminus and expressed in E.coli BL21 (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. ELISA method was used to detect its binding activity to anti-DR5 monoclonal antibody (mAb) mDRA-6.


RESULTS
Human DR5 extracellular fragment gene was successfully amplified and high level expression was obtained in E.coli BL21 (DE3) induced by 0.1 mmol/L IPTG. The DR5 extracellular fragment protein was identified by SDS-PAGE and Western blot analysis. ELISA results showed that the purified DR5 could be recognized by mDRA-6.


CONCLUSIONS
The extracellular region of DR5 with bioactivity has been successfully expressed and purified, which lay the foundation for further study.
Chen
Ju-gao
JG
Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475001, China.
Wang
Yu-gang
YG
Zhao
Kun-peng
KP
Gu
Xin
X
Li
Yan
Y
Ma
Yuan-fang
YF
Shen
Bei-fen
BF
D017209:Apoptosis; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D012995:Solubility; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism