N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage.
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.
Watanabe
Takayoshi
T
School of Materials Science, Japan, Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Miyata
Yoichi
Y
Abe
Ryoji
R
Muranaka
Norihito
N
Hohsaka
Takahiro
T
D000143:Acids / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry; D002474:Cell-Free System; D004952:Esters / Q000737:chemistry* / Q000378:metabolism*; D006900:Hydroxylation; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000143:Acids; D000596:Amino Acids; D004952:Esters; D008164:Luminescent Proteins; D019809:Streptavidin