β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions
β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions
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DOI：
10.1038/nbt0602-619
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发表时间：
2002-06-01
影响因子：
46.9
通讯作者：
Michnick, SW
中科院分区：
文献类型：
--
作者：
Galarneau, A;Primeau, M;Michnick, SW
我们之前描述了一种检测蛋白质-蛋白质相互作用的策略，该策略基于合理设计的酶片段的蛋白质相互作用辅助折叠。我们将此策略称为蛋白质片段互补测定 (PCA) 1-5。在这里，我们描述了基于酶 TEM-1 β-内酰胺酶 (EC: 3.5.2.6) 的 PCA，其中包括使用头孢菌素硝基头孢菌素进行的简单比色体外测定，以及使用荧光底物 CCF2/AM 在完整细胞中进行的测定（参考文献 6）。使用细胞裂解物在体外测定中测试了 GCN4 亮氨酸拉链和凋亡蛋白 Bcl2 和 Bad 的组成型蛋白-蛋白相互作用，以及 Smad3 的同二聚化。通过相同的体外测定，我们还证明了蛋白激酶 PKB 与底物 Bad 的相互作用。体外测定方法简便，适合高通量筛选模式，信号背景比在 10:1 至 250:1 范围内，优于迄今为止开发的其他 PCA。此外，我们表明体外测定可用于小分子诱导的蛋白质相互作用、雷帕霉素诱导的 FKBP 和酵母 FRB（TOR 的 FKBP-雷帕霉素结合域（雷帕霉素靶标））的相互作用的定量分析。该测定再现了已知的解离常数和这种相互作用的位点数量。哺乳动物细胞中β-内酰胺酶的体外比色和体内荧光测定的结合表明了多种灵敏和高通量的大规模应用，包括蛋白质-蛋白质或酶-蛋白质相互作用的体外蛋白质阵列分析和体内应用，例如表达相互作用蛋白质伴侣的细胞的克隆选择。
We have previously described a strategy for detecting protein-protein interactions based on protein interaction-assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA) 1-5. Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein-protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10: 1 to 250: 1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule-induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein-protein or enzyme-protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.