An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore
SCIE
SCOPUS
- Title
- An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore
- Authors
- Liu, JQ; Hanne, J; Britton, BM; Shoffner, M; Albers, AE; Bennett, J; Zatezalo, R; Barfield, R; Rabuka, D; Lee, JB; Fishel, R
- Date Issued
- 2015-11-19
- Publisher
- Nature Publishing
- Abstract
- Fluorophore labeling of proteins while preserving native functions is essential for bulk Forster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E. coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/13437
- DOI
- 10.1038/SREP16883
- ISSN
- 2045-2322
- Article Type
- Article
- Citation
- SCIENTIFIC REPORTS, vol. 5, 2015-11-19
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