High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites.
| Autor: | Ozawa K; Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. ozawa@rsc.anu.edu.au, Loscha KV, Kuppan KV, Loh CT, Dixon NE, Otting G |
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| Jazyk: | angličtina |
| Zdroj: | Biochemical and biophysical research communications [Biochem Biophys Res Commun] 2012 Feb 24; Vol. 418 (4), pp. 652-6. Date of Electronic Publication: 2012 Jan 24. |
| DOI: | 10.1016/j.bbrc.2012.01.069 |
| Abstrakt: | Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract. (Copyright © 2012 Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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