Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags
Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags
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DOI：
10.2144/00283st02
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发表时间：
2000-03-01
期刊：
影响因子：
2.7
通讯作者：
Ustav, M
中科院分区：
文献类型：
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作者：
Kaldalu, N;Lepik, D;Ustav, M
我们在此描述了将两个新定位的牛乳头瘤病毒1型（BPV - I）E2蛋白表位用作标签的情况。我们构建了几种载体质粒，用于在大肠杆菌或真核细胞中过表达以及适度表达单标签或双标签蛋白。新标签与几种蛋白质融合，并且在不同的检测中测试了带标签蛋白质的活性。结果表明，这些标签在体内和体外都不会干扰这些蛋白质的功能。单克隆抗体3F12和1E2与其各自的表位M的相互作用具有特异性，并且在多种条件下具有高亲和力。我们已经证明，3F12抗体 - 表位相互作用可耐受高达2M的高盐浓度。这使得能够在高盐缓冲液中对带标签的蛋白质进行免疫沉淀和免疫纯化，并减少污染蛋白质的非特异性结合。我们还提供了一种使用带标签的树脂结合DNA结合蛋白进行DNA结合和DNA酶I足迹分析的实验方案。牛乳头瘤病毒I型E2衍生的标签可被推荐为用于蛋白质检测和纯化的有用工具。
We describe here the use of two newly mapped bovine papillomavirus type I (BPV-I) E2 protein epitopes as rags. We constucted several vector plasmids for overexpression as Ic ell as for moderate expression of single- or double-tugged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tugged proteins was tested in different assays. The rags were shown nor to interfere, with the function of these proteins in I vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes M as specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations rip to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged resin-bound DNA-binding proteins. The BPV-I E2-derived tags can be recommended as useful tools for detection and purification of proteins.