N-terminal modification increases the stability of the recombinant human endostatin in vitro
N-terminal modification increases the stability of the recombinant human endostatin in vitro
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DOI：
10.1042/ba20090063
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发表时间：
2009-10-01
影响因子：
2.8
通讯作者：
Chen, Yali
中科院分区：
文献类型：
--
作者：
Jiang, Li-Ping;Zou, Chang;Chen, Yali
Endostar是一种人血管内皮抑制素的衍生物，在其N-末端修饰了一个额外的金属螯合序列（MGGSHHHHH），被中国国家食品药品监督管理局批准用于治疗非小细胞肺癌。这种修饰有助于内皮抑制素序列中的额外锌结合位点。在本研究中，锌结合和无锌的endostar进行了比较，以进一步表征其生化和结构特性。通过监测荧光发射光谱的变化来确定热诱导变性。数据表明，锌结合显着增加的转变温度的恩度，并有助于在蛋白质构象可逆的变化后，再冷却。蛋白质水解实验表明，修饰蛋白与锌离子结合后，在胰蛋白酶、胰凝乳蛋白酶和羧肽酶A和B的作用下，能稳定恩度的N-末端和C-末端。Western-blot分析表明，用胰蛋白酶和糜蛋白酶处理后，Endostar的主要裂解片段位于N-末端。在人脐静脉内皮细胞的增殖试验中，具有额外锌结合位点的锌结合和无锌Endostar样品显示出相似的抑制活性。
Endostar, approved for the treatment of non-small-cell lung cancer by the State Food and Drug Administration in China, is a derivative of human endostatin that is modified with an additional metal-chelating sequence (MGGSHHHHH) at the N-terminus. This modification contributes to an additional zinc-binding site in the endostatin sequence. In the present study, zinc-binding and zinc-free endostar were compared to further characterize their biochemical and structural properties. Thermally induced denaturation was determined by monitoring changes in fluorescence emission spectra. The data indicated that zinc binding significantly increased the transition temperature of endostar and contributed to a reversible change in protein conformation after recooling. Proteolysis assays demonstrated that the modified protein binding with zinc ions can stabilize the N-terminus and the C-terminus of endostar when treated with trypsin, chymotrypsin and carboxypeptidase A and B. Western-blot analyses using anti-His, antibody confirmed that the major cleaved fragments of endostar were in the N-terminus when treated with trypsin and chymotrypsin. In the proliferation assay with human umbilical-vein endothelial cells, the zinc-binding and zinc-free endostar samples with extra zinc-binding sites displayed similar inhibiting activities.