STTR Phase I: An On-Demand Protein Engineering Platform

这个小企业技术转让（STTR）项目的更广泛的影响/商业潜力将是开发高通量蛋白质表达的平台技术。目前用于表达蛋白质组的标准涉及广泛的生物信息学、克隆、体内表达和测定。这种方法需要不同领域的专业知识，以及数周到数月的时间才能成功执行。此外，由于毒性或纯化困难，可能难以表达复杂蛋白质，需要对表达和纯化条件进行劳动密集型诊断。所提出的平台允许通过提供组合的离体计算、表达和测定系统以显著的成本和时间节省表征数百种蛋白质序列。这允许快速访问生物数据和按需蛋白质序列原型。作为该平台的一部分开发的方法也将允许K-12和本科生更多地接触生物工程，需要很少的资金或先前的生物学经验。通过降低蛋白质工程的成本和时间，并在一个简单的系统中工作，不需要生物信息学，克隆，细胞培养和生化表征的知识，生物学家和非生物学家都将能够进行相关的生物工程研究和快速测试蛋白质设计假设。该STTR第一阶段项目提议开发一个高通量和计算辅助平台，以快速收集不同蛋白质组的生化数据。使用这个平台，研究人员将能够从单个参考蛋白质中表达数百种相关蛋白质变体。产率为微摩尔值，每次运行提供高达50 ug/50 uL。 因此，可以产生足够的蛋白质用于详细的生物化学表征和活性测定。所提出的平台是一个包罗万象的离体计算，表达和测定系统。 在这个项目中，细胞色素P450酶的工程和原型设计，重要的工业和制药催化剂，将被证明与结束第二阶段的目标，原型1,000不同的细胞色素P450酶从设计到表征在不到一个星期。

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project will be the development of a platform technology for high-throughput protein expression. The current standard for expressing panels of proteins involves extensive bioinformatics, cloning, in vivo expression, and assays. This method takes significant expertise in disparate fields, and weeks to months of time to perform successfully. Furthermore, it can be difficult to express complex proteins due to toxicity or purification difficulty, requiring labor-intensive diagnosis of expression and purification conditions. The proposed platform allows characterization of hundreds of protein sequences at significant cost and time savings by providing a combined ex vivo computational, expression, and assay system. This allows rapid access to biological data, and on-demand protein sequence prototyping. The methods developed as part of this platform also will allow greater access to biological engineering for K-12 and undergraduate students, requiring little capital or prior biological experience. By reducing costs and time for protein engineering, and by working in a simple system that requires no knowledge of bioinformatics, cloning, cell culturing, and biochemical characterization, biologists and non-biologists alike will be able to conduct relevant biological engineering research and rapidly test protein design hypotheses. This STTR Phase I project proposes to develop a high-throughput and computationally assisted platform to rapidly collect biochemical data on a diverse set of proteins. Using this platform, researchers will be able to conduct expression of hundreds of relevant protein variants from a single reference protein. The yields are micromolar-values, providing up to 50ug/50uL per run. Therefore, enough protein can be generated for detailed biochemical characterization and activity assays. The proposed platform is an all-encompassing ex-vivo computational, expression, and assay system. In this project, engineering and prototyping of cytochrome P450 enzymes, important industrial and pharmaceutical catalysts, will be demonstrated with an end-Phase II goal to prototype 1,000 diverse cytochrome P450 enzymes from design to characterization in less than a week.