STTR Phase I: An On-Demand Protein Engineering Platform

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.