Tom Irene, Estevez Alberto, Bowman Krista, Gonzalez Lino C
Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
Department of Structural Biology, Genentech, South San Francisco, CA 94080, USA.
Anal Biochem. 2015 Jun 15;479:1-5. doi: 10.1016/j.ab.2015.03.015. Epub 2015 Mar 20.
When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors.
当与多价蛋白质探针结合使用时，蛋白质微阵列提供了一种强大的技术，用于发现低亲和力的细胞外蛋白质-蛋白质相互作用。用于受体匹配筛选的探针通常由与亲和标签融合的纯化细胞外结构域组成。鉴于大约三分之二的细胞外蛋白质是含有跨膜结构域的蛋白质，开发一种在类似天然膜环境中表达和展示探针受体的系统将是很有必要的。为此，我们评估了杆状病毒展示作为一种为蛋白质微阵列筛选生成多价探针的平台。生成了展示单跨膜结构域受体BTLA、CD200和EFNB2的病毒粒子，这些受体对其相互作用伴侣具有一系列亲和力。用Cy5荧光团直接标记的病毒粒子与包含600多种细胞外蛋白质的微阵列进行筛选，并将结果与来自可溶性Fc蛋白或探针包被的蛋白A微珠的数据进行比较。采用与不相关的表达探针的对照杆状病毒进行封闭步骤的优化方案，能够识别出预期的相互作用，其信噪比与其他形式获得的信噪比相似或更高。我们的结果表明，杆状病毒展示适用于检测蛋白质微阵列上的高亲和力和低亲和力细胞外蛋白质-蛋白质相互作用。该平台无需蛋白质纯化，并为膜相关受体提供了类似天然的脂质环境。
