Infoscience

Conference paper

Protein chemistry in living cells

Studying proteins in living cells generally requires the construction of a fusion protein between the protein of interest and an appropriate tag. Currently used fusion protein-based assays rely on the genetically encoded properties of the tag, which significantly limits their scope of applications. Here we present a general method for the in vivo labeling of fusion proteins with small mols. possessing functionalities that can not be genetically encoded. The specific attachment of the small mol. to the fusion protein is based on the unusual mechanism of the human DNA repair protein O6-alkylguanine DNA alkyltransferase (hAGT), which irreversibly transfers the alkyl group from its substrate, O6-alkylguanine, to one of its cysteine residues. The approach can be used to label fusion proteins with different fluorophores, affinity labels and small mols. able to control transcription. We also demonstrate how directed evolution of hAGT can be used to increase the efficiency of the in vivo labeling. [on SciFinder (R)]

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