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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44GM086930-03
Application #
8496825
Study Section
Special Emphasis Panel (ZRG1-IMST-G (10))
Program Officer
Maas, Stefan
Project Start
2009-02-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$420,705
Indirect Cost
Name
New England Biolabs, Inc.
Department
Type
DUNS #
066605403
City
Ipswich
State
MA
Country
United States
Zip Code
01938
Tuckey, Corinna; Asahara, Haruichi; Zhou, Ying et al. (2014) Protein synthesis using a reconstituted cell-free system. Curr Protoc Mol Biol 108:16.31.1-16.31.22
Chong, Shaorong (2014) Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. Curr Protoc Mol Biol 108:16.30.1-16.30.11