Prokaryotic expression and functional study of human high mobility group box 1 protein
- VernacularTitle:人高迁移率族蛋白1的原核细胞表达及其功能研究
- Author:
Lei ZHAO
;
Jinghua LIU
;
Jing TANG
;
Zhijie LI
;
Yawei LIU
;
Peng DENG
;
Yong JIANG
- Publication Type:Journal Article
- Keywords:
High mobility group proteins;
Prokaryotic expression;
Cytokines;
Umbilical vein endothelial cells
- From:
Chinese Journal of Pathophysiology
1989;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To construct the prokaryotic expression plasmid of His-tagged human high mobility group box 1 fusion protein (hHMGB1) and to express the fusion protein in E. coli for the affinity purification. METHODS: The cDNA coding region of HMGB1 was amplified by PCR from pGEX4T-HMGB1 and cloned into a modified pET14b vector following the routine procedure. After identification by enzyme digestion, PCR and sequencing, the plasmid was transformed into BL21 (DE_3) competent cells, and the His-HMGB1 fusion protein was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), and further purified by Ni-NTA affinity chromatography. The protein was filtered for sterilization and used to stimulate human umbilical vein endothelial cells (HUVECs). 24 hours later, the cultured supernatant of HUVECs was collected for the detection of cytokines/chemokines with LiquiChip system. RESULTS: The His-HMGB1 fusion protein expression plasmid was identified by enzyme digestion and sequencing. The purified His-tagged fusion protein was analyzed by SDS-PAGE and Western blotting with specific anti-His antibody. It was found that the production of IL-8 from HUVECs was highly induced in a dose-dependent manner by HMGB1. CONCLUSION: The His-tagged HMGB1 fusion protein expression plasmid was successfully constructed, and purified. Recombinant HMGB1 protein has a high bioactivity on the induction of cytokines in HUVECs, which may significantly facilitate the future study of HMGB1 biological functions.
