Expression,purification and identification of the human high mobility group-1 protein code gene in E.coli
Objective To construct the recombinant vector, the expression of human HMGB1 code gene and to study its function. Methods Human HMGB1 code gene was amplified by RT-PCR and cloned into vector pGEM-T easy. Then it was subcloned into prokaryotic expressive vector pGEX4T-2. After four hours induction by IPTG, the expression of recombinant 56kDa fusion protein GST-HMGB1 was identified by SDS-PAGE. Purified HMGB1 was eluted from GSTrap FF affinity chromatography after thrombin cleavage. To identified the function of purified proteins, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pGEX-HMGB1 was constructed. The 30kDa purified proteins were obtained. The recombinant HMGB1 stimulated THP1 to secret TNF-α and enhanced the apoptosis of THP1 cells. Conclusion The purified prokaryotic expression human HMGB1 were obtained. It might be important for study of the function of human HMGB1.