Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

Kai Johnsson, Antje Keppler, Horst Pick, Horst Vogel
2004
Article

Résumé

The in vivo and in vitro labeling of fusion proteins with synthetic mols. capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivs. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivs. and the use of such derivs. for the labeling of different hAGT fusion proteins in vivo and in vitro. [on SciFinder (R)]

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https://infoscience.epfl.ch/record/81373?ln=fr

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A general method for the covalent labeling of fusion proteins with small molecules in vivo

Thomas Gronemeyer, Kai Johnsson, Antje Keppler, Horst Pick, Horst Vogel

Characterizing the movement, interactions, and chem. microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic mols. capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small mol. to a protein, the formation of stable complexes between biarsenical compds. and peptides contg. cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

2003

Chargement

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We report here the generation of mutants of the human O6-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter mols. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O6-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O6-benzylguanine derivs., the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small mols. and therefore should become important tools for studying protein function in living cells. In addn. to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O6-benzylguanine. [on SciFinder (R)]

2003

Chargement