Production and characterization of ubiquitin proteins

描述（由申请人提供）：定义良好的新型泛素衍生物将是基础研究和药物发现工作的宝贵工具，但这些在商业上不可用。我们建议在I期SBIR拨款中开发一个全面的泛素变体小组，包括赖氨酸突变和n端修饰的所有组合，以及n端表位和半抗原标签的集合。每个泛素将在E1激活和E2结合试验中进行能力测试。未修饰和修饰的泛素蛋白面板将用于使用各种泛素偶联酶系统产生具有独特赖氨酸键的非锚定多泛素链。将开发体外合成、放大、纯化和表征交替链键的方法。这些多泛素链底物将在相关的和新的生化分析中广泛表征。最后将测试它们对一系列去泛素化酶的水解，包括异肽酶T、UCH-L1、UCH-L3、BAP1和USP14，以表征这些酶的水解特异性。一旦完全表征，这些新的泛素蛋白和多泛素链将在II期商业化，并作为独立试剂提供给整个研究界，并作为设计任何去泛素化酶抑制剂的更广泛的分析平台的一部分。这项工作还将产生利用综合连接和修饰类型在商业规模上生产泛素链的能力。这个定义的产品套件为新的研究工具和分析创造了宝贵的选择，这将促进和加速发现和目标研究工作。这样一个蛋白质小组将彻底改变泛素相关研究的速度和难度，促进对错误或异常泛素化过程的基本问题的进一步理解，并有助于发现新的治疗方法，特别是在去泛素化和蛋白酶体方面。

DESCRIPTION (provided by applicant): Well-defined and novel ubiquitin derivatives would be invaluable tools for basic research and drug discovery efforts, but these are commercially unavailable. We propose in this Phase I SBIR grant to develop a comprehensive panel of ubiquitin variants, making all combinations of lysine mutations and N-terminal modifications, as well as a collection of N-terminal epitope and hapten tags. Each ubiquitin will be tested for competence in E1 activation and E2 conjugation assays. The panel of unmodified and modified ubiquitin proteins will be used to produce unanchored polyubiquitin chains with unique lysine linkages, using various ubiquitin conjugation enzymes systems. Methods to synthesize, scale-up, purify to homogeneity and characterize alternate chain linkages in vitro will be developed. These polyubiquitin chain substrates will be extensively characterized in relevant and novel biochemical assays. They will finally be tested for hydrolysis against a battery of deubiquitinating enzymes, including Isopeptidase T, UCH-L1, UCH-L3, BAP1, and USP14, to characterize these hydrolytic specificities of these enzymes. Once fully characterized, these novel Ubiquitin proteins and polyubiquitin chains will be commercialized in Phase II and made available to the entire research community as both free standing reagents, and as part of a broader assay platform for designing inhibitors of any deubiquitination enzyme. This work will also result in the ability to produce, at commercial scale, ubiquitin chains using comprehensive linkages and modification types. This defined product suite creates invaluable options for new research tools and assays, which will facilitate and accelerate discovery and target research efforts. Such a panel of proteins would revolutionize the speed and ease of ubiquitin-related research endeavors, facilitate the further understanding of fundamental questions about misdirected or abnormal ubiquitination processes, and aid in the discovery of new therapeutics, especially as regards to deubiquitination and the proteasome.