[Gene expression and primary functional characterization of human DR5 extracellular fragment]

To acquire human DR5 extracellular fragment with bioactivity.

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.

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.

The extracellular region of DR5 with bioactivity has been successfully expressed and purified, which lay the foundation for further study.