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Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist.

Biochemistry. Biokhimii͡a

metabolism, Animals, HMGB1 Protein, antagonists & inhibitors, genetics, Mice, Polymyxin B, chemistry, Protein Folding, Cell Line, Protein Structure, Tertiary, Recombinant Fusion Proteins, isolation & purification, Thrombomodulin, Tumor Necrosis Factor-alpha

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      Abstract

      High mobility group box chromosomal protein 1 (HMGB1) is a lethal mediator of systemic inflammation, and its A box domain is isolated as an antagonist of HMGB1. To enhance its expression level and its anti-HMGB1 effect, the A box cDNA was coupled with the sequence encoding lectin-like domain of thrombomodulin (TMD1). The fusion DNA fragment was ligated into the prokaryotic expression vector pQE-80L to construct the recombinant plasmid pQE80L-A/TMD1. The plasmid was then transformed into Escherichia coli DH5alpha, and the recombinant fusion protein A/TMD1 was expressed at 37 degrees C for 4 h, with induction by IPTG at the final concentration of 0.2 mM. The expression level of the fusion protein was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA chromatography and the purity was about 95%. After passing over a polymyxin B column to remove any contaminating lipopolysaccharides, the purified protein was tested for its anti-inflammatory activity. Our data show that A/TMD1 significantly inhibits HMGB1-induced TNF-alpha release and might be useful in treating HMGB1-elevated sepsis.

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      20618136

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