Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity.
Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity.
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DOI：
10.1016/j.bbrc.2006.05.031
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发表时间：
2006-07
影响因子：
3.1
通讯作者：
D. Wei;Yan Gao;Xiang-rong Cao;N. Zhu;Jianhui Liang;W. Xie;M. Zhen;Min Zhu
中科院分区：
文献类型：
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作者：
D. Wei;Yan Gao;Xiang-rong Cao;N. Zhu;Jianhui Liang;W. Xie;M. Zhen;Min Zhu
内皮抑素的生物活性、复性和多聚体形成，特别是从细菌中产生的重组内皮抑素，被证明是临床应用的挑战。为了检测重组内皮抑素多聚体的生物学活性，我们首先在大肠杆菌中表达了内皮抑素，并通过离子交换层析纯化了内皮抑素。纯化的活性蛋白可以自发地诱导多聚体形成，但仍具有相当的活性。目的研究内皮抑素多聚体的抗血管生成活性，采用反相高效液相色谱法成功分离内皮抑素单体和多聚体，用于抗血管生成活性的测定。绒毛尿囊膜（CAM）抑制实验结果表明，单体和多聚体均能显著抑制CAM血管化。在0.8μg剂量下，多聚体蛋白和单体蛋白的抑制率分别为58%和38%。多聚体内皮抑素比单体内皮抑素具有更高的活性（p<0.05）。当蛋白质浓度低于0.4μg/ml时，两者的抑制率均无显著性差异（p>0.05）。HUVEC增殖实验结果显示，0.6和1.6μg/ml的多聚体对HUVEC增殖的抑制作用与单体相似，多聚体抑制HUVEC增殖的活性高于单体（p<0.05）。总之，我们的研究结果表明，内皮抑素多聚体具有相当或更高的生物活性和多聚化不会影响其生物活性，这意味着内皮抑素活性是不敏感的结构构象贡献的二硫键。
The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8μg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p<0.05). However, when the protein dosage is less than 0.4μg/ml, there is no significance between their inhibition rates (p>0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6μg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p<0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds.