Viala Julie P M, Bouveret Emmanuelle
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, UMR7255, Institut de Microbiologie de la Méditerranée, Aix-Marseille University-CNRS, 31 Chemin Joseph Aiguier, 13009, Marseille, France.
Methods Mol Biol. 2017;1615:221-232. doi: 10.1007/978-1-4939-7033-9_18.
The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.
蛋白质-蛋白质相互作用网络的发现能够揭示形成细胞机器的蛋白质复合物，或揭示特定细胞途径的组成蛋白。因此，解析蛋白质-蛋白质相互作用网络有助于更深入地理解细胞的功能。在此，我们描述了在细菌中进行串联亲和纯化（TAP）的实验方案，该方案能够在天然条件下鉴定诱饵蛋白的相互作用伙伴。此方法包括使用两种不同标签进行的两个连续亲和纯化步骤。为此，将诱饵蛋白与TAP标签进行翻译融合，TAP标签由钙调蛋白结合肽（CBP）和金黄色葡萄球菌蛋白A（ProtA）的两个免疫球蛋白G（IgG）结合结构域组成，这两个结构域由烟草蚀纹病毒（TEV）蛋白酶切割位点隔开。在基于ProtA与包被IgG的珠子结合进行第一轮纯化后，TEV蛋白酶切割释放出带有CBP标签的诱饵蛋白及其相互作用伙伴，以便在钙调蛋白亲和树脂上进行第二轮纯化，而与IgG结合的蛋白质污染物则被留在后面。在染色体位点创建TAP标签翻译融合能够检测在生理条件下发生的蛋白质相互作用。
