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N‐Terminal Specific Fluorescence Labeling of Proteins through Incorporation of Fluorescent Hydroxy Acid and Subsequent Ester Cleavage
Author(s) -
Watanabe Takayoshi,
Miyata Yoichi,
Abe Ryoji,
Muranaka Norihito,
Hohsaka Takahiro
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700578
Subject(s) - cleavage (geology) , fluorescence , chemistry , biochemistry , amino acid , green fluorescent protein , peptide , immunoprecipitation , biology , gene , paleontology , physics , quantum mechanics , fracture (geology)
Abstract We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four‐base codon‐mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell‐free translation system. We found that a fluorescent‐labeled p ‐amino‐ L ‐phenyllactic acid was successfully incorporated downstream of N‐terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N‐terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N‐terminal specific labeling of proteins with various compounds.