An efficient system for high-level expression and easy purification of authentic recombinant proteins
An efficient system for high-level expression and easy purification of authentic recombinant proteins
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DOI：
10.1110/ps.04618904
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发表时间：
2004-05-01
期刊：
影响因子：
8
通讯作者：
Baker, RT
中科院分区：
文献类型：
--
作者：
Catanzariti, AM;Soboleva, TA;Baker, RT
已发现重组蛋白作为与真核蛋白泛素的融合物的表达显著增加不稳定或表达差的蛋白的产率。该技术的益处通过天然存在的去泛素化酶的可用性进一步增强，所述酶从融合产物中去除泛素。然而，由于缺乏一个强大的，容易纯化的去泛素化酶的系统的通用性受到限制。在这里，我们报告了一个有效的表达系统的发展，利用泛素融合技术，它允许方便的高产量和易于纯化的真实蛋白。构建大肠杆菌载体（pHUE）用于表达组氨酸标记的泛素融合蛋白，并表达和纯化组氨酸标记的去泛素化酶来切割这些融合蛋白。使用大小和复杂性不同的几种蛋白质测试表达系统。这些结果表明，该方法将适用于广泛的蛋白质和肽的表达和快速纯化，并且应该适合于高通量应用。
Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.