Ecole Polytechnique Federale De Lausanne

Ecole Polytechnique Féderale De Lausanne (epfl)

Antje Keppler

Kai Peter Johnsson

Alexandre Juillerat

Hughes Jaccard

Christian Heinis

Thomas Gronemeyer

Susanne Gendreizig

Jan Barnikow

Susanne Kindermann (née Gendreizig)

Christopher Chidley

Kai Johnsson

A method of using O-alkylguanine-DNA alkyltransferase (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.

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A method using O-alkylguanine-DNA alkyltransferases (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.

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 O-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N-substituted O-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.

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Heidelberg, Germany

Antje Keppler