Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist
Expression and purification of the fusion protein HMGB1Abox-TMD1, a novel HMGB1 antagonist
复制标题
新型 HMGB1 拮抗剂融合蛋白 HMGB1Abox-TMD1 的表达和纯化
DOI：
10.1134/s0006297910040103
复制
发表时间：
2010-04-01
影响因子：
2.8
通讯作者：
He, Fengtian
中科院分区：
文献类型：
--
作者：
Li, Yuan;Gong, Wei;He, Fengtian
高迁移率族盒染色体蛋白 1 (HMGB1) 是全身炎症的致命介质，其 A 盒结构域作为 HMGB1 的拮抗剂被分离出来。为了增强其表达水平及其抗 HMGB1 效果，将 A 盒 cDNA 与编码血栓调节蛋白 (TMD1) 凝集素样结构域的序列偶联。将融合DNA片段连接至原核表达载体pQE-80L中，构建重组质粒pQE80L-A/TMD1。将质粒转化大肠杆菌DH5α，37℃表达重组融合蛋白A/TMD1 4 h，IPTG诱导，终浓度0.2 mM。融合蛋白的表达量高达细胞总蛋白的40%。融合蛋白经Ni-NTA层析纯化，纯度约为95%。通过多粘菌素 B 柱去除任何污染的脂多糖后，测试纯化的蛋白质的抗炎活性。我们的数据表明，A/TMD1 显着抑制 HMGB1 诱导的 TNF-α 释放，可能有助于治疗 HMGB1 升高的脓毒症。
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 intoEscherichia coliDH5α, 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.