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Fluorescent protein resonance energy transfer for high-throughput screening.

机译：用于高通量筛选的荧光蛋白共振能量转移。

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The desire to track proteins and monitor their functions inside of living cells has driven the development of new tools and techniques capable of making these measurements. Fluorescent protein Forster resonance energy transfer (FRET) is an attractive choice for probing intracellular events, but the low efficiency of most available pairs limits the utility of the technique. To address this problem, a quantitative evolutionary strategy using fluorescence-activated cell sorting was applied to optimize a cyan-yellow fluorescent protein pair for FRET. Multiple rounds of mutation, followed by sorting of the leading edge of the cell population enabled step-wise evolution of variants with improved FRET properties. The best pair of variants, CyPet-YPet, exhibited a seven-fold improvement in dynamic range over the parental pair when measured in vitro. Additionally, the optimized pair provided significantly improved resolution of FRET signal changes in bacterial, yeast, and mammalian cells using flow cytometry. The improvements in dynamic range were verified in applications including the detection of protease activities, protein-protein interactions, and small molecules.; The improved utility of CyPet and YPet enabled flow cytometric sorting on the basis of fluorescent protein FRET for the first time. Clones expressing active caspase-3 were isolated from inactive background present in 10 5-fold excess using the FRET signal change as an indicator of protease function. This proof of principle experiment paves the way for new opportunities in the isolation and evolution of potentially therapeutic proteases. Additionally, a FRET-based protein-protein interaction screen was developed, and enabled enrichment of peptide binders to an SH3 domain and a PDZ2 domain from large libraries. Consensus sequences determined from isolated peptides matched the known consensus binding motifs for these targets. This FRET approach may have advantages over yeast two-hybrid and other screens. Additionally, flow cytometric sorting techniques developed for isolating rare cells on the basis of small FRET signal changes may have more widespread applicability to situations where target cell fluorescence differs subtly from that of nontarget cells. These tools and techniques developed for fluorescent protein FRET applications create opportunities for expansion of the utility of FRET in the discovery and study of biological interactions and activities.
机译：跟踪蛋白质并监测其在活细胞中的功能的需求推动了能够进行这些测量的新工具和技术的发展。荧光蛋白Forster共振能量转移（FRET）是探测细胞内事件的诱人选择，但是大多数可用对的低效率限制了该技术的实用性。为了解决这个问题，应用了荧​​光激活细胞分选的定量进化策略来优化FRET的青黄色荧光蛋白对。多轮突变，然后对细胞群的前沿进行排序，使得逐步改善了FRET特性的变体得以进化。最好的一对变体CyPet-YPet在体外测量时，其动态范围比亲本对表现出七倍的改善。此外，使用流式细胞仪，优化的对可显着提高细菌，酵母和哺乳动物细胞中FRET信号变化的分辨率。动态范围的改善已在包括蛋白酶活性，蛋白质-蛋白质相互作用和小分子检测在内的应用中得到验证。 CyPet和YPet的改进效用首次使基于荧光蛋白FRET的流式细胞仪分类成为可能。使用FRET信号变化作为蛋白酶功能的指标，从以10 5倍过量存在的非活性背景中分离出表达活性caspase-3的克隆。这项原理性实验证明为潜在的治疗性蛋白酶的分离和进化提供了新的机会。此外，开发了基于FRET的蛋白质-蛋白质相互作用筛选，并能够将肽结合剂从大型文库富集至SH3域和PDZ2域。由分离的肽确定的共有序列与这些靶标的已知共有结合基序匹配。这种FRET方法可能比酵母双杂交和其他筛选方法具有优势。另外，开发用于基于小的FRET信号变化来分离稀有细胞的流式细胞仪分选技术可能更广泛地应用于靶细胞荧光与非靶细胞荧光微妙差异的情况。为荧光蛋白FRET应用而开发的这些工具和技术为扩展FRET在生物相互作用和活性的发现和研究中的用途创造了机会。

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