Expression and Purification of the Fusion Protein HMGB1Abox-TMD1, a Novel HMGB1 Antagonist

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Yuan Li^#, Wei Gong^#, Li Zhang, Gang Huang, Jialin Li, Xiaodong Shen, and Fengtian He^*

Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, China; fax: +86(23)6875-3462; E-mail: hefengtian06@yahoo.com.cn

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received May 25, 2009; Revision received October 9, 2009

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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 DH5α, and the recombinant fusion protein A/TMD1 was expressed at 37°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-α release and might be useful in treating HMGB1-elevated sepsis.

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KEY WORDS: fusion protein, high mobility group box chromosomal protein 1, thrombomodulin

DOI: 10.1134/S0006297910040103

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