High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins
High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins
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DOI：
10.1006/prep.2001.1434
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发表时间：
2001-07-01
影响因子：
1.6
通讯作者：
Acharya, KR
中科院分区：
文献类型：
--
作者：
Holloway, DE;Hares, MC;Acharya, KR
血管生成素(Angigenin，Ang)是胰腺核糖核酸酶超家族的一种小分子碱性蛋白，能有效地诱导新生血管的形成，是一种很有前途的抗癌靶点。小鼠具有编码血管生成素的一个同源基因(Mang)和三个同源基因，分别命名为血管生成素相关蛋白(MAngrp)、血管生成素-3(mang-3)和血管生成素-4(mang-4)。现有的异源表达系统中蛋白质产量低，阻碍了对这些同源物的结构和功能的研究。在所描述的实验中，我们使用pET表达载体在大肠杆菌BL21-CodonPlus(DE3)-RIL细胞的细胞质中表达这些蛋白，然后每个蛋白以不溶性聚集体的形式大量积累。这些蛋白用精氨酸辅助复性，然后用阳离子交换层析和反相高效液相色谱纯化，每个纯化的蛋白都对tRNA具有酶活性，纯mAngrp和MANG-3的产量分别为7.6 mg/L和12 mg/L培养物，比以前报道的实验有很大的提高。这也是首次报道MANG-4的表达和纯化，其产量为30 mg/L培养物。这些蛋白质毫克量的现成供应将使进一步的功能研究和高分辨率结构分析得以进行。(C)2001年学术出版社。
Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily, It potently induces the formation of new blood vessels and has emerged as a promising anticancer target. Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4). Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system. In the experiments described, we used a pET expression vector to express these proteins in the cyto plasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates. The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA, The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments. This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture. The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted.(C) 2001 Academic Press.