title: An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.
abstract: Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster 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.
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Jiaquan
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Jeungphill
authors.initials: J
authors.lastname: Hanne
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Brooke M
authors.initials: BM
authors.lastname: Britton
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Matthew
authors.initials: M
authors.lastname: Shoffner
authors.affiliation: Catalent Biologics-West, Emeryville, CA 94608.
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Albers
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Jared
authors.initials: J
authors.lastname: Bennett
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.
authors.forename: Rachel
authors.initials: R
authors.lastname: Zatezalo
authors.affiliation: Catalent Biologics-West, Emeryville, CA 94608.
authors.forename: Robyn
authors.initials: R
authors.lastname: Barfield
authors.affiliation: Catalent Biologics-West, Emeryville, CA 94608.
authors.forename: David
authors.initials: D
authors.lastname: Rabuka
authors.affiliation: Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, Korea.|School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea.
authors.forename: Jong-Bong
authors.initials: JB
authors.lastname: Lee
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH 43210.|Physics Department, The Ohio State University, Columbus, OH 43210.
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli