Premium
Optimized Fluorescent Trimethoprim Derivatives for in vivo Protein Labeling
Author(s) -
Calloway Nathaniel T.,
Choob Michael,
Sanz Ana,
Sheetz Michael P.,
Miller Lawrence W.,
Cornish Virginia W.
Publication year - 2007
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.200600414
Subject(s) - green fluorescent protein , fluorescence , dihydrofolate reductase , fluorescence microscope , fluorescent protein , biochemistry , cell sorting , microscopy , chemistry , in vivo , fluorescent labelling , biophysics , cell free protein synthesis , protein tag , chinese hamster ovary cell , recombinant dna , protein biosynthesis , biology , in vitro , fusion protein , enzyme , gene , physics , microbiology and biotechnology , quantum mechanics , optics , receptor
The combined technologies of optical microscopy and selective probes allow for real‐time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein‐targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild‐type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal‐to‐noise ratio by using fluorescence microscopy and fluorescence‐activated cell sorting (FACS). We also show that fluorescent TMP–eDHFR complexes are complements to green fluorescent protein (GFP) for two‐color protein labeling experiments in cells.