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

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.).
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 Record created 2006-02-27, last modified 2018-03-18

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