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首页> 外文期刊>Protein Expression and Purification >One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system
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One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

机译：使用多顺反子表达系统一步一步从细菌包涵体中纯化全长重组人AP-1复合物的亲和标签

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摘要

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun x 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. (C) 2008 Elsevier Inc. All rights reserved.
机译：AP-1转录因子是主要在Jun（c-Jun，JunB，JunD）和Fos（c-Fos，FosB，Fra-1，Fra-2）家族成员之间形成的二聚体蛋白复合物。这些不同的AP-1复合物在许多细胞类型中表达，并调节与细胞增殖，分化和应激反应有关的靶基因表达。尽管早已认识到AP-1的重要性，但AP-1的生化特性仍然受到限制，部分原因是难以纯化具有活性DNA结合和转录活性的全长，重组二聚体。使用细菌共表达和表位标记方法的组合，我们成功地纯化了全长人AP-1复合物的所有12种异二聚体（3 Jun x 4 Fos）以及c-Jun / c-Jun，JunD / JunD和共表达AP-1亚基变性和复性后，使用一步镍-NTA亲和标签纯化法从细菌包涵体得到c-Jun / JunD二聚体。与细菌中单个蛋白质的表达相比，二聚AP-1复合物中两个组成成分的共表达有助于稳定蛋白质。纯化的二聚体AP-1复合物在序列特异性DNA结合中具有功能，如电泳迁移率迁移分析和DNase I足迹所示，并且在含有AP-1结合物的体外重组人乳头瘤病毒（HPV）染色质中也具有转录活性。 HPV核小体天然结构中的位点。这些重组全长人AP-1复合物的可用性极大地促进了许多生物系统中AP-1调控的基因转录的机理研究。 （C）2008 Elsevier Inc.保留所有权利。

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