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Trypsiligase‐Catalyzed Labeling of Proteins on Living Cells
Author(s) -
Liebscher Sandra,
Mathea Sebastian,
Aumüller Tobias,
Pech Andreas,
Bordusa Frank
Publication year - 2021
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.202000718
Subject(s) - fluorescence , transmembrane protein , chemistry , biochemistry , folding (dsp implementation) , membrane protein , dipeptide , fusion protein , peptide , function (biology) , covalent bond , rhodamine , green fluorescent protein , biophysics , membrane , biology , microbiology and biotechnology , receptor , recombinant dna , gene , physics , organic chemistry , quantum mechanics , electrical engineering , engineering
Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site‐directed linkage of small‐sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg‐His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.