Link, A. James and Vink, Mandy K. S. and Agard, Nicholas J. and Prescher, Jennifer A. and Bertozzi, Carolyn R. and Tirrell, David A. (2006) Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proceedings of the National Academy of Sciences of the United States of America, 103 (27). pp. 10180-10185. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:LINpnas06
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Abstract
The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 -> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.
| Item Type: | Article |
|---|---|
| Additional Information: | Copyright © 2006 by the National Academy of Sciences Edited by Dieter Söll, Yale University, New Haven, CT, and approved April 28, 2006 (received for review February 10, 2006). Published online before print June 26, 2006, 10.1073/pnas.0601167103 We thank H. Jakubowski (New Jersey Medical School, Newark, NJ) and Y. Mechulam (École Polytechnique, Paliseau, France) for the generous donation of plasmids encoding MetRS. This work was supported by National Institutes of Health Grants GM58867 (to C.R.B.) and GM62523 (to D.A.T.) and by the U.S. Army Research Office through Institute for Collaborative Biotechnologies Grant DAAD19-03-D-0004. Author contributions: A.J.L., C.R.B., and D.A.T. designed research; A.J.L., M.K.S.V., N.J.A., and J.A.P. performed research; A.J.L., M.K.S.V., N.J.A., J.A.P., C.R.B., and D.A.T. analyzed data; and A.J.L. and D.A.T. wrote the paper. Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. |
| Subject Keywords: | azide–alkyne ligation; azidohomoalanine; azidonorleucine; click chemistry |
| Record Number: | CaltechAUTHORS:LINpnas06 |
| Persistent URL: | http://resolver.caltech.edu/CaltechAUTHORS:LINpnas06 |
| Alternative URL: | http://dx.doi.org/10.1073/pnas.0601167103 |
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. |
| ID Code: | 6880 |
| Collection: | CaltechAUTHORS |
| Deposited By: | Archive Administrator |
| Deposited On: | 30 Dec 2006 |
| Last Modified: | 26 Dec 2012 09:26 |
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