Construction of recombinant prokaryotic expression plasmid of human high mobility group box1 and its expression and purification

Objective To construct the prokaryotic expression plasmid of human high mobility group box1 (hHMGB1) gene,and to express and purify the His-tagged hHMGB1 fusion protein. Methods According to the sequence reported in GenBank,we optimized codons by OptimumGeneTM and synthesized the human HMGB1 gene. To construct prokaryotic expression plasmid pQE-T7-2 /HMGB1,the DNA fragment obtained from PCR was digested with NdeⅠ and Xho Ⅰ,and then inserted into pQE-T7-2 that was cut with the same enzymes. The recombinants were screened by colony PCR,restriction mapping and sequencing. The recombinant E. coli BL21 (DE3) was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG),and the recombinant protein was identified by SDS-PAGE and Western blotting. The recombinant human HMGB1 was further purified by Ni-NTA Agarose. Results The prokaryotic expression plasmid pQE-T7-2 /HMGB1 was successfully constructed. The expressed recombinant protein accounted for about 20% of the total bacterial proteins. The recombinant protein could specifically react with anti-HMGB1 antibody and anti-His antibody. The purity of recombinant human HMGB1 separated with affinity chromatography was more than 90% . Conclusion The recombinant prokaryotic expression vector of human HMGB1 which was successfully constructed may lay the foundation for the further study of the function of human HMGB1.