Mutants of o6-alkylguanine-dna alkyltransferase

The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus (c) improved expression yield as soluble protein and improved stability in various hosts (d) improved stability under oxidising conditions (e) improved stability within cells after reaction with a substrate (f) improved stability outside cells before and after reaction with a substrate (g) improved in vitro solubility (h) improved reactivity against 0-alkylguanine substrates (1) reduced reactivity against DNA-based substrates and (j) reduced reactivity against N-substituted 0-alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally I to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest.

Mutants of human O6-alkylguanine-DNA alkyltransferase with improved properties

Christopher Chidley, Thomas Gronemeyer, Christian Heinis, Kai Johnsson, Alexandre Juillerat, Antje Keppler

The invention relates to human O6-alkylguanine-DNA alkyltransferase (hAGT) mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localization of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as sol. protein and improved stability in various hosts; (d) improved stability under oxidizing conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro soly.; (h) improved reactivity against O6-alkylguanine substrates; (i) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted 06-alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally 1 to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest. [on SciFinder (R)]

(EPFL Ecole Polytechnique Federale de Lausanne, Switz.).2005

Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

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

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

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Christian Heinis, Hugues Jaccard, Kai Johnsson, Alexandre Juillerat, Béatrice Kunz, India Christina Sielaff

Fusion proteins of human O6-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-mol. inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells. [on SciFinder (R)]

2005