CELL-FREE PROTEIN TRANSLATION AND FP BINDING AFFINITIES

人类基因组计划、组合化学和测定微型化已经产生了对更快、均质方法来表征蛋白质/蛋白质相互作用的需求。该第一阶段应用提出使用无细胞翻译荧光标记蛋白质，使用肽标签亲和纯化蛋白质，并使用荧光偏振测量结合亲和力。含有荧光氨基酸的错酰化tRNA将用于在特定琥珀（UAG）位点标记蛋白质，从而控制标记的位置和频率。标记化学将针对荧光偏振结果和翻译期间非天然氨基酸的有效掺入进行优化。最终，这些方法可用于药物发现，用于筛选，特异性测试，结构/功能研究，以及更好地了解治疗药物靶向途径。拟议的商业应用：快速生产荧光蛋白探针的能力将大大提高PanVera开发用于药物发现的HTS检测的能力。该技术以试剂盒形式商业化，用于探测蛋白质-蛋白质相互作用，也将在研究产品市场产生可观的收入。

The human genome project, combinatorial chemistry, and assay miniaturization have created a demand for faster, homogeneous methods to characterize protein/protein interactions. This Phase I application proposes to fluorescently label proteins using cell-free translation, affinity purify the proteins using peptide tags, and measure binding affinities using fluorescence polarization. Misacylated tRNA's containing fluorescent amino acids will be used to label proteins at specific amber (UAG) sites, thereby controlling the location and the frequency of labeling. The labeling chemistry will be optimized for fluorescence polarization results and for efficient incorporation of the unnatural amino acid during translation. Ultimately, these methods could be used in drug discovery for screening, specificity testing, structure/function studies, and developing a better understanding of therapeutic drug target pathways. PROPOSED COMMERCIAL APPLICATIONS: The ability to rapidly produce fluorescent protein probes will greatly enhance PanVera's capability to develop HTS assays for drug discovery. Commercialization of the technology in a kit format for probing protein - protein interactions should also generate significant revenue in the research products market.