Cloning, expression and purification of functionally active human angiopoietin-like protein 2.
Cloning, expression and purification of functionally active human angiopoietin-like protein 2.
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DOI：
10.1186/2193-1801-3-337
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Allen BG
中科院分区：
文献类型：
--
作者：
Farhat N;Mamarbachi AM;Thorin E;Allen BG
血管生成素样蛋白2(Angptl2)是一种分泌型糖蛋白，与血管生成、炎症和动脉粥样硬化有关，并可增强人造血干细胞的存活。Angptl2的糖基化是生物活性所必需的，而血管生成素样蛋白2的研究由于缺乏成熟形式的这种蛋白的来源而受到阻碍。我们描述了一种系统，该系统允许从稳定转染的HEK 293细胞的条件培养液中纯化人Angptl2的糖基化形式。为了在纯化的同时保持Angptl2‘S内源性N端分泌信号肽的完整性，在Angptl2编码序列的下游融合了GST。分泌型Angptl2-GST的纯化采用一步谷胱甘肽亲和纯化方案。用SDS-PAGE、免疫印迹和质谱仪对产物的纯度和纯度进行了鉴定。N-糖苷酶处理降低了Angptl2-GST在SDS-PAGE上的表观分子质量，证实了其糖基化状态。纯化的人Angptl2-GST在体外刺激HUVEC迁移和微管形成。所获得的Angptl2-GST的产量适合于多种用途，包括体外和体内的功能分析。
Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-like protein 2 have been hindered by the lack of a source for the mature form of this protein. We describe a system that permits purification of the glycosylated form of human Angptl2 from conditioned media of stably transfected HEK 293 cells. To facilitate purification while retaining the integrity of Angptl2’s endogenous N-terminal secretion signal peptide, GST was fused downstream of the Angptl2 coding sequence. Secreted Angptl2-GST was purified using a one-step glutathione-affinity purification scheme. The purity and identity of the resulting protein were confirmed by SDS-PAGE, immunoblotting, and mass spectrometry. N-Glycosidase treatment reduced the apparent molecular mass of Angptl2-GST on SDS-PAGE, confirming its glycosylation state. Purified human Angptl2-GST stimulated both HUVEC migration and microtubule formation in vitro. The yield of Angptl2-GST obtained was in quantities suitable for multiple applications including functional in vitro and in vivo assays.
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影响因子：
82.9
作者：
Zhang, CC;Kaba, M;Lodish, HF
通讯作者：
Lodish, HF
影响因子：
64.8
作者：
Zheng, Junke;Umikawa, Masato;Cui, Changhao;Li, Jiyuan;Chen, Xiaoli;Zhang, Chaozheng;Hyunh, HoangDinh;Kang, Xunlei;Silvany, Robert;Wan, Xuan;Ye, Jingxiao;Canto, Alberto Puig;Chen, Shu-Hsia;Wang, Huan-You;Ward, E. Sally;Zhang, Cheng Cheng
通讯作者：
Zhang, Cheng Cheng
影响因子：
4
作者：
Bernatchez, PN;Tremblay, F;Sirois, MG
通讯作者：
Sirois, MG
影响因子：
4.8
作者：
Camenisch, G;Pisabarro, MT;Gerber, HP
通讯作者：
Gerber, HP
DOI：
10.1073/pnas.0501902102
发表时间：
2005-09-20
影响因子：
11.1
作者：
Kubota, Y;Oike, Y;Suda, T
通讯作者：
Suda, T