A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells
A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells
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DOI：
10.1016/j.pep.2005.03.035
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发表时间：
2005-07-01
影响因子：
1.6
通讯作者：
Cáceres, JF
中科院分区：
文献类型：
--
作者：
Cazalla, D;Sanford, JR;Cáceres, JF
在这里，我们描述了一种在哺乳动物细胞中表达和纯化活性重组蛋白的简单有效的方法。该方法首先在瞬时转染的293T细胞中表达T7表位标记蛋白，然后进行抗T7琼脂糖亲和层析。这个过程产生大约75和100 μ g的生物活性蛋白/150厘米(2)烧瓶，可用于生化研究。我们对原型SR蛋白SF2/ASF的表达进行了测试，SF2/ASF是SR蛋白家族的一员，在组成剪接和选择性剪接中起作用。我们发现，使用该方法纯化的SF2/ASF能够补充S100 HeLa提取物，证明其具有生物活性。此外，一种新的sr相关蛋白的表达，它是前mrna剪接的第二步所必需的，也产生了一个活性蛋白。总之，我们提出了一种基于哺乳动物细胞瞬时转染的方案，可以很容易地纯化大量的生物活性蛋白。(c) 2005爱思唯尔公司版权所有。
Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 mu g of biologically active protein/150cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins. (c) 2005 Elsevier Inc. All rights reserved.