Mutants of O 6 -alkylguanine-DNA alkyltransferase

Details Mutants of O 6 -alkylguanine-DNA alkyltransferase Mutants of O 6 -alkylguanine-DNA alkyltransferase

2011-02-15

2011-02-15

Pak, Yong D (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S440000, C435S069100, C435S071100, C536S023200

Reexamination Certificate

active

07888090

ABSTRACT:
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 O6-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted O6-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.

REFERENCES:
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M. Xu-Welliver et al., “Point Mutations at Multiple Sites Including Highly Conserved Amino Acids Maintain Activity, but Render O-Benzylguanine”, Biochemical Journal, vol. 347, No. 2, pp. 519-526, Apr. 15, 2000.

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