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A New Strategy for the Site-Specific Modification of Proteins in Vivo.
- Source :
-
Biochemistry . 6/10/2003, Vol. 42 Issue 22, p6735. 12p. 4 Color Photographs, 5 Black and White Photographs, 2 Diagrams, 1 Chart, 3 Graphs. - Publication Year :
- 2003
-
Abstract
- We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-L-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-L-phenylalanine or p-acetyl-L-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of > 75%. In specific examples, m-acetyl-L-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo. [ABSTRACT FROM AUTHOR]
- Subjects :
- *PROTEIN engineering
*BACTERIAL proteins
*ESCHERICHIA coli
Subjects
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 42
- Issue :
- 22
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10037251
- Full Text :
- https://doi.org/10.1021/bi0300231