CaltechAUTHORS
  A Caltech Library Service

Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase

Kiick, Kristi L. and Tirrell, David A. (2000) Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase. Tetrahedron, 56 (48). pp. 9487-9493. ISSN 0040-4020. http://resolver.caltech.edu/CaltechAUTHORS:KIIt2000

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:KIIt2000

Abstract

The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The incorporation of amino acids into proteins in vivo is controlled in large part by the aminoacyl-tRNA synthetases (AARS). We have measured kinetic constants for in vitro activation of a set of methionine analogues by methionyl-tRNA synthetase (MetRS) via the ATP–PPi exchange reaction. Activation of methionine analogues in vitro correlates well with the ability of these analogues to support protein synthesis in vivo, substantiating the critical role of the AARS in controlling the incorporation of non-natural amino acids into proteins. Methionine analogues with k_(cat)/K_m values 2000-fold lower than those for methionine can support synthesis of a typical target protein (mDHFR) under standard conditions of protein expression. The kinetic constants correlate well with observed protein yields from a conventional bacterial expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Furthermore, increasing the MetRS activity of the bacterial host results in increased protein synthesis in media supplemented with the methionine analogues homoallylglycine and norleucine. These results suggest new strategies for incorporation of non-natural amino acids via manipulation of the AARS activity of a bacterial host.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/S0040-4020(00)00833-4DOIArticle
http://www.sciencedirect.com/science/article/pii/S0040402000008334PublisherArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2000 Elsevier. Received 22 May 2000; accepted 2 August 2000. This work was supported by grants from the Polymers and Genetics Programs of the U.S. National Science Foundation and from the U.S. Army Research Office. We are grateful to J. C. M. van Hest, R. Weberskirch, and H. E. Schoemaker for synthesis of the methionine analogues, to H. Jakubowski for donation of the plasmid pGG3, and to R. Alexander for helpful discussions regarding activation assays. K. L. K thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship.
Funders:
Funding AgencyGrant Number
NSFUNSPECIFIED
Army Research Office (ARO)UNSPECIFIED
National Defense Science and Engineering Graduate (NDSEG) FellowshipUNSPECIFIED
Subject Keywords:aminoacyl-tRNA synthetases; biosynthesis; kinetics; polymers; in vivo protein engineering; non-natural amino acids
Record Number:CaltechAUTHORS:KIIt2000
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:KIIt2000
Official Citation:Kristi L. Kiick, David A. Tirrell, Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase, Tetrahedron, Volume 56, Issue 48, 24 November 2000, Pages 9487-9493, ISSN 0040-4020, http://dx.doi.org/10.1016/S0040-4020(00)00833-4. (http://www.sciencedirect.com/science/article/pii/S0040402000008334)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53360
Collection:CaltechAUTHORS
Deposited By: Anne Hormann
Deposited On:06 Feb 2015 22:56
Last Modified:06 Feb 2015 22:56

Repository Staff Only: item control page