NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
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DOI：
10.1021/acschembio.5b00753
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发表时间：
2016-02-01
影响因子：
4
通讯作者：
Wood, Keith V.
中科院分区：
文献类型：
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作者：
Dixon, Andrew S.;Schwinn, Marie K.;Wood, Keith V.
蛋白质片段互补分析（PCA）被广泛用于研究蛋白质相互作用。然而，使用的片段在结构上受到损害，并且尚未优化或彻底表征以准确评估这些相互作用。我们利用NanoLuc的小尺寸和明亮的发光来设计新的互补报告基因（NanoBiT）。通过设计，NanoBiT亚基（即，1.3 kDa肽，18 kDa多肽）弱缔合，使得它们组装成发光复合物由它们所附接的靶蛋白的相互作用特征决定。为了确定它们用于测量相互作用亲和力和动力学的一般适用性，我们确定它们的固有亲和力（K-D = 190 μ M）和缔合常数（k（on）= 500 M-1 s（-1），k（off）= 0.2 s（-1））在蛋白质相互作用的典型范围之外。使用先前表征的SME-1 β-内酰胺酶与一组抑制剂结合蛋白之间的相互作用，在规定的生化条件下验证了NatioBiT的准确性。在细胞中，NanoBiT与FRB/FKBP的融合产生了与NanoLuc的线性特征一致的发光。使用蛋白激酶A和β-抑制蛋白-2评估的响应动力学是快速的、可逆的，并且对温度（21-37摄氏度）是稳健的。最后，NanoBiT提供了一种方法来测量已知诱导BRAF和CRAF之间相互作用的激酶抑制剂的药理学。我们的研究结果表明，NanoBiT的内在特性允许蛋白质相互作用的准确表示，报告可靠，动态地在细胞中响应。
Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (K-D = 190 mu M) and association constants (k(on) = 500 M-1 s(-1), k(off) = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NatioBiT was, verified tinder defined biochemical conditions using the previously characterized interaction between SME-1 beta-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and beta-arrestin-2, were rapid, reversible, and robust to temperature (21-37 degrees C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.