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首页> 外文期刊>Protein Expression and Purification >Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: A useful tool for studying interactions between Ets-1 and its partners
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Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: A useful tool for studying interactions between Ets-1 and its partners

机译：在大肠杆菌中生物素化重组Ets-1生产的高效系统：研究Ets-1及其合作伙伴之间相互作用的有用工具

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Identification of Ets-1 interaction partners is critical for understanding its properties. Ets-1 DNA-binding is governed by an intramolecular mechanism called autoinhibition. Ets-1 increases its DNA-binding affinity by counteracting autoinhibition through binding either to a particular organization of Ets binding sites (EBS) in palindrome, as in the Stromelysin-1 promoter, or to EBS adjacent to DNA-binding sites of its partners by combinatorial interactions, as in the Collagenase-1 promoter. Identification of new Ets-1 interaction partners should allow the identification of new functions for this transcription factor. To this end, we fused a biotin tag to Ets-1 protein in order to copurify it and its partners by affinity. For the first time, we cloned, produced in Escherichia coli and purified a biotinylated recombinant Ets-1 protein using the T7-Impact (TM) system (New England Biolabs (R)), adapted to induce biotinylation. Nearly 100% biotinylation was attained without altering Ets-1 properties. Biotinylated Ets-1 bound to and transactivated the Stromelysin-1 promoter the same way as native Ets-1 did. It also conserved interactions with known Ets-1 partners such as c-Jun, Erk-2 and Runx-1. In addition, streptavidin pull-down and surface plasmon resonance assays demonstrated that biotinylated Ets-1 is a useful tool for qualitative and quantitative studies of Ets-1 interaction with its partners. (C) 2008 Elsevier Inc. All rights reserved.
机译：Ets-1相互作用伙伴的识别对于理解其特性至关重要。 Ets-1 DNA的结合受称为自动抑制的分子内机制支配。 Ets-1通过与回文中特定结构的Ets结合位点（EBS）结合（如Stromelysin-1启动子）或与其伴侣的DNA结合位点相邻的EBS结合来抵消自身抑制作用，从而提高了其DNA结合亲和力。组合相互作用，如Collagenase-1启动子。新的Ets-1相互作用伙伴的鉴定应允许鉴定该转录因子的新功能。为此，我们将生物素标签融合到Ets-1蛋白上，以通过亲和力共纯化它及其伴侣。我们首次克隆，在大肠杆菌中生产并使用适于诱导生物素化的T7-Impact（TM）系统（New England Biolabs（R））纯化了生物素化的重组Ets-1蛋白。在不改变Ets-1性质的情况下获得了近100％的生物素化。生物素化的Ets-1以与天然Ets-1相同的方式与Stromelysin-1启动子结合并使其活化。它还保留了与已知的Ets-1合作伙伴（例如c-Jun，Erk-2和Runx-1）的互动。此外，抗生蛋白链菌素的下拉和表面等离振子共振分析表明，生物素化的Ets-1是定性和定量研究Ets-1及其伴侣的相互作用的有用工具。 （C）2008 Elsevier Inc.保留所有权利。

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