Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist.
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.
Li
Yuan
Y
Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing, 400038, China.
Gong
Wei
W
Zhang
Li
L
Huang
Gang
G
Li
Jialin
J
Shen
Xiaodong
X
He
Fengtian
F
D000818:Animals; D002460:Cell Line; D024243:HMGB1 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011112:Polymyxin B / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D024243:HMGB1 Protein; D011993:Recombinant Fusion Proteins; D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha; D011112:Polymyxin B