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.
Highlights
Cys residue to an fGly produces a reactive aldehyde that may be used for chemical coupling[25,26,27]
These results suggested that the high concentrations of hydrazide-dyes induced solution instability of EcMutS and that hydrazide-fluorophore labeling of the ald6-tagged MMR protein was largely non-specific
Our results indicate that high expression of FGE may reduce the expression of an ald6-tagged protein in E.coli, while low expression of FGE can lead to incomplete fGly conversion and reduced labeling efficiency
Summary
Cys residue to an fGly produces a reactive aldehyde that may be used for chemical coupling[25,26,27]. Co-expression of FGE with LCTPSR-containing target proteins appeared to catalyze Cys → fGly conversion in vivo permitting chemical coupling of a hydrazide-modified fluorophore[1]. Substantial fluorophore labeling was reported a number of technical issues arose that included: (1) the use of large quantities of expensive hydrazide-modified dyes (75.6 mM; 60 mg Cy3/ml) to obtain extensive labeling, (2) the conversion of Cys to fGly in vivo was not quantified, (3) the specificity of fluorophore labeling to the fGly residue was not determined, and (4) the effects of the labeling process on overall protein specific-activity was not fully determined. The protocol relies on efficient and controlled FGE conversion of Cys to fGly in vitro followed by specific and irreversible fluorophore labeling using the Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation method. The portability of the FGE recognition sequence should make HIPS-fluorophore labeling widely applicable for single molecule imaging experiments as well as bulk and kinetic FRET interaction studies
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