A new strategy for the site-specific modification of proteins in vivo

被引:167
作者
Zhang, ZW
Smith, BAC
Wang, L
Brock, A
Cho, C
Schultz, PG
机构
[1] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA
[2] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
[3] Sidney Kimmel Canc Ctr, San Diego, CA 92121 USA
[4] Novartis Res Fdn, Gen Inst, San Diego, CA 92121 USA
关键词
D O I
10.1021/bi0300231
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-Lphenylalanine 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.
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页码:6735 / 6746
页数:12
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