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Site‐Specific Dual Labeling of Proteins by Using Small Orthogonal Tags at Neutral pH
Author(s) -
Grünewald Jan,
Jones David H.,
Brock Ansgar,
Chiu HsienPo,
Bursulaya Badry,
Ng Kenneth,
Vo Todd,
Patterson Paula,
Uno Tetsuo,
Hunt James,
Spraggon Glen,
Geierstanger Bernhard H.
Publication year - 2014
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.201402204
Subject(s) - conjugate , chemistry , förster resonance energy transfer , lysine , peptide , biochemistry , protein array analysis , fluorescence , combinatorial chemistry , green fluorescent protein , biophysics , computational biology , biology , amino acid , gene expression , gene , dna microarray , mathematical analysis , physics , mathematics , quantum mechanics
To expand the utility of proteinaceous FRET biosensors, we have developed a dual‐labeling approach based on two small bio‐orthogonal tags: pyrroline‐carboxy‐lysine (Pcl) and the S6 peptide. The lack of cross‐reactivity between those tags enables site‐specific two‐color protein conjugation in a one‐pot reaction. Moreover, Pcl/S6 dual‐tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE‐Fc, respectively. Both proteins could be efficiently dual‐labeled with FRET‐compatible fluorescent dyes at neutral pH. In the case of IgE‐Fc, the resulting conjugate enabled the monitoring of IgE binding to its high‐affinity receptor FcεRI, which is a key event in allergic disease.