Ecole Polytechnique Federale De Lausanne

Epfl Ecole Polytechnique Fédérale De Lausanne

Hughes Jaccard

Kai Peter Johnsson

Alexandre Juillerat

Maik Kindermann

Antje Keppler

Christian Heinis

Thomas Gronemeyer

India Christina Sielaff

Jan Barnikow

Christopher Chidley

Kai Johnsson

The invention relates to substrates for O-alkylguanine-DNA alkyltransferases (AGT) of formula R-A-X&#x2014;CH&#x2014;R&#x2014;R-L, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, Ris a group &#x2014;R-Lor a group R, Rand Rare, independently of each other, a linker, Ris an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH, Ris arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, Lis a label, a plurality of same or different labels, a bond connecting Rto A forming a cyclic substrate, or a further group &#x2014;R&#x2014;CH&#x2014;X-A-R, and Lis a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.

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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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Yverdon-les-Bains, Switzerland

Hughes Jaccard