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Site-Specific Dual-Labeling of Proteins Using Small Orthogonal Tags at Neutral pH

Grunewald, Jan and Jones, David and Brock, Ansgar and Bursulaya, Badry and Patterson, Paula and Ng, Kenneth and Vo, Michael and Uno, Tetsuo and Hunt, James and Spraggon, Glen and Geierstanger, Bernhard (2014) Site-Specific Dual-Labeling of Proteins Using Small Orthogonal Tags at Neutral pH. ChemBiochem.

Abstract

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, FcRI, which is considered a key event in allergic reactions.

Item Type: Article
Date Deposited: 26 Apr 2016 23:45
Last Modified: 26 Apr 2016 23:45
URI: https://oak.novartis.com/id/eprint/21617

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