Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions.
Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions.
复制标题
DOI：
10.3791/3881
复制
发表时间：
2012-03-05
期刊：
影响因子：
--
通讯作者：
Wright GJ
中科院分区：
文献类型：
--
作者：
Kerr JS;Wright GJ
胞外蛋白：分泌或膜系蛋白之间的蛋白质相互作用对于启动细胞间通讯和确保多细胞生物内的凝聚力都至关重要。预测形成细胞外相互作用的蛋白质由大约四分之一的人类基因1编码，但尽管它们的重要性和丰度，这些蛋白质中的大多数没有记录的结合伴侣。首先，这是由于它们的生物化学难处理性：膜包埋的蛋白质难以溶解在其天然构象，并含有结构上重要的翻译后修饰。此外，受体蛋白之间的相互作用亲和力通常以极低的相互作用强度（半衰期< 1秒）为特征，从而排除了许多常用的高通量方法2对其的检测。 在这里，我们描述了一种检测方法，AVEXIS（基于亲和力的细胞外相互作用筛选），它克服了这些技术挑战，能够以低假阳性率检测非常弱的蛋白质相互作用（t1/2 ≤ 0.1秒）3。该测定通常以高通量形式实施，以便能够以方便的微量滴定板形式系统地筛选数千种相互作用（图1）。它依赖于可溶性重组蛋白文库的产生，所述可溶性重组蛋白文库含有细胞表面受体或分泌蛋白的胞外域片段，在所述胞外域片段内筛选相互作用;因此，该方法适用于I型、II型、GPI连接的细胞表面受体和分泌蛋白，但不适用于多通道膜蛋白，如离子通道或转运蛋白。重组蛋白文库使用方便的高水平哺乳动物表达系统4生产，以确保添加重要的翻译后修饰，如糖基化和二硫键。表达的重组蛋白分泌到培养基中，并以两种形式产生：生物素化诱饵（可捕获在适于筛选的链霉亲和素包被的固相上）和五聚酶标记（β-内酰胺酶）猎物。诱饵蛋白和猎物蛋白以二元方式相互呈递，以检测它们之间的直接相互作用，类似于常规ELISA（图1）。通过来自软骨寡聚基质蛋白（COMP）的肽序列实现猎物中蛋白质的五聚化，并增加胞外域的局部浓度，从而提供显著的亲合力增益，从而能够检测到甚至非常短暂的相互作用。通过在筛选前将诱饵和猎物的活性标准化至预定水平，我们已经表明，可以以低假阳性率3检测到单体半衰期为0.1秒的相互作用。
Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods2. Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 ≤ 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters. The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (β-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.
登录
查看更多内容
影响因子：
4.8
作者：
Dustin, ML;Golan, DE;van der Merwe, PA
通讯作者：
van der Merwe, PA
影响因子：
7
作者：
Bushell, K. Mark;Sollner, Christian;Wright, Gavin J.
通讯作者：
Wright, Gavin J.
影响因子：
6.4
作者：
Jiang, Lei;Barclay, A. Neil
通讯作者：
Barclay, A. Neil
富含神经亮氨酸重复受体的细胞表面相互作用网络。
影响因子：
12.3
作者：
Söllner C;Wright GJ
通讯作者：
Wright GJ
DOI：
10.1074/mcp.m110.004119
发表时间：
2010-12
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Martin S;Söllner C;Charoensawan V;Adryan B;Thisse B;Thisse C;Teichmann S;Wright GJ
通讯作者：
Wright GJ