期刊
JOURNAL OF BIOTECHNOLOGY
卷 178, 期 -, 页码 12-22出版社
ELSEVIER
DOI: 10.1016/j.jbiotec.2014.02.009
关键词
Residue-specific incorporation; Unnatural amino acids; Streptavidin; Cell-free protein synthesis; Tryptophan
资金
- National Security Science and Engineering Faculty Fellowship [FA9550-10-1-0169]
- Welch Foundation [F-1654]
- National Science Foundation-Sandpit [MCB-0943383]
- postdoctoral fellowship from the Cancer Prevention and Research Institute of Texas
- Applied Research Laboratories at The University of Texas at Austin
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0943383] Funding Source: National Science Foundation
Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. (c) 2014 Elsevier B.V. All rights reserved.
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