STTR Phase I: An On-Demand Protein Engineering Platform

这个小型企业技术转移(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。目前的蛋白质表达标准包括广泛的生物信息学、克隆、体内表达和分析。这种方法需要在不同领域的大量专业知识，并需要几周到几个月的时间才能成功执行。此外，由于毒性或纯化困难，可能难以表达复杂的蛋白质，需要对表达和纯化条件进行劳动密集型诊断。建议的平台通过提供一个组合的体外计算、表达和分析系统，以显著的成本和时间节省了数百个蛋白质序列的特征。这允许快速访问生物数据，并按需制作蛋白质序列原型。作为这一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，只需要很少的资金或以前的生物学经验。通过减少蛋白质工程的成本和时间，并在不需要生物信息学、克隆、细胞培养和生化特性知识的简单系统中工作，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，并快速测试蛋白质设计假说。这个STTR第一阶段项目建议开发一个高通量和计算辅助的平台，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进行数百个相关蛋白质变体的表达。产率为微摩尔值，每次提供高达50ug/50uL。因此，可以产生足够的蛋白质来进行详细的生化表征和活性分析。建议的平台是一个包罗万象的体外计算、表达和检测系统。在这个项目中，将展示重要的工业和制药催化剂细胞色素P450酶的工程和原型，目标是在不到一周的时间内，在不到一周的时间内制作1000种不同的细胞色素P450酶的原型。

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.