Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation
The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.
© 2002 by the National Academy of Sciences Communicated by Ralph F. Hirschmann, University of Pennsylvania, Philadelphia, PA, October 31, 2001 (received for review August 15, 2001). Published online before print December 18, 2001, 10.1073/pnas.012583299 We acknowledge the generous donation of plasmids encoding MetRS from H. Jakubowski and Y. Mechulam and the assistance of J. Kua in modeling the azide-functionalized amino acids. This research was supported by the Office of Naval Research, Grant N00014–98-1–0605 and Order N00014–98-F-0402 through the U.S. Department of Energy under Contract DE-AC03–76SF00098, the National Institutes of Health (GM58867–01), the Polymers and Genetics Programs of the U.S. National Science Foundation, and the U.S. Army Research Office. K.L.K. thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship. E.S. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. The Center for New Directions in Organic Synthesis is supported by Bristol-Myers Squibb as a supporting member. K.L.K. and E.S. contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Published - KIIpnas02.pdf