SBIR-STTR Award

Directed protein evolution is a powerful technology for generating proteins with desired properties, such as thermostability. There is currently no effective in vitro method to select proteins with enhanced thermostability. Thermostable proteins have a wide range of industrial and medical applications. The aim of this project is to construct a reconstituted in vitro protein synthesis system (thermoPURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC. The thermoPURE system may allow protein synthesis to be conducted in vitro at elevated temperatures, and due to its low background activities, facilitate in vitro screening and selection or other applications that require elevated temperatures. In conjunction with powerful in vitro selection technologies such as in vitro compartmentalization and ribosome display, the thermoPURE may become the only system that allows in vitro directed evolution of thermostable proteins from their mesophilic origins. During the initial phase of the project, we will clone a complete set of genes for the translation factors, aminoacyl-tRNA synthetases and energy regeneration enzymes from Thermus thermophilus. All proteins will be overexpressed and purified from E. coli. The ribosomes and tRNAs will be purified from a growing culture of Thermus thermophilus. Using our experience and capacity of manufacturing the E. coli version of the reconstituted protein synthesis system (the PURE system), we will reconstitute the protein translation machinery of Thermus thermophilus and demonstrate in vitro protein synthesis at elevated temperatures. In the second phase, we will apply the thermoPURE system for in vitro evolution of mesophilic proteins such as restriction enzymes, RNA polymerases, and single-chain antibodies with an ultimate goal to generate valuable proteins with enhanced thermostability for industrial and medical usage.

Public Health Relevance:
Proteins that are stable at elevated temperatures have a wide range of industrial and medical applications. There is currently no effective in vitro method to select proteins with enhanced thermostability. The goal of this project is to provide such a method by constructing a reconstituted in vitro protein synthesis system from purified components of Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC.

Public Health Relevance:
Proteins that are stable at elevated temperatures have a wide range of industrial and medical applications. There is currently no effective in vitro method to select proteins with enhanced thermostability. The goal of this project is to provide such a method by constructing a reconstituted in vitro protein synthesis system from purified components of Thermus thermophilus, a bacterium that grows at an optima temperature of 720C.

Thesaurus Terms:
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Directed protein evolution is a powerful technology for protein engineering. It generally involves generating a library of protein variants and identifying those with desired properties by selection or screening. In terms of library complexity, in vitro selection methods are superior to cell-based selection methods by several orders of magnitude, allowing a much larger sequence space in proteins to be sampled. There is currently no effective in vitro method to select proteins with desired mutations that enhance their thermostability. Thermostable proteins can function at high temperatures, are generally robust and resistant to degradation under a variety of conditions, and therefore are valuable for a wide range of industrial and medical applications. This project intends to develop a technology for in vitro selection of protein thermostability. We plan to achieve this goal by (1) constructing a reconstituted in vitro protein synthesis system (thermo PURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 720C, and (2) applying such system for directed evolution of proteins with enhanced thermostability. We have successfully completed the first phase of the project (Phase I) and established an initial thermo PURE system that allowed in vitro synthesis of active full- length proteins at temperatures up to 600C. For the Phase II, we propose to commercialize the thermo PURE system by optimizing the system and testing more target proteins. We also propose to use the thermo PURE system in conjunction with in vitro selection technologies, such as in vitro compartmentalization and ribosome display, for directed evolution of thermostable proteins from their mesophilic origins. We plan to test several selection schemes of directed evolution for a variety of enzymes and proteins. If successful, this project would lead to the following unique and valuable commercial products: (1) a protein thermo synthesis kit;(2) a service for engineering proteins with enhanced thermostability;(3) thermostable nucleic acid enzymes as new reagents for research communities and thermostable single-chain antibodies for therapeutic applications.

Public Health Relevance:
Proteins can be engineered to function at high temperatures and exhibit robustness and resistance to degradation under a variety of conditions. This project intends to develop a technology to select engineered thermostable proteins for industrial and medical use.