Methods using O6-alkylguanine-DNA alkyltransferases

Kai Johnsson, Antje Keppler
(Ecole Polytechnique Federale De Lausanne, Switz.)., 2002
Brevet

Résumé

A method using O6-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 mols. to the fusion proteins that introduce a new phys. or chem. property to the fusion protein. Examples of such mols. are, among others, spectroscopic probes or reporter mols., affinity tags, mols. generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or mols. suitable for the immobilization of the fusion protein. A peptide-human AGT fusion protein was labeled in Escherichia coli using I. Biotinylated proteins were detected by Western blotting. [on SciFinder (R)]

Official source

https://infoscience.epfl.ch/record/81345?ln=fr

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Methods using o-alkylguanine-dna alkyltransferases

Kai Johnsson, Antje Keppler

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.

2004

Chargement

O6-alkylguanine-DNA alkyltransferases for efficient labeling of fusion proteins

Kai Johnsson, Alexandre Juillerat, Antje Keppler

The present invention relates to methods of transferring a label from suitable substrates to O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins, to suitable fusion proteins, to suitable variants of AGT, and to novel labeled fusion proteins obtained. A protein of interest is incorporated into an AGT fusion protein, the AGT fusion protein is contacted with an AGT substrate carrying a label, and the AGT fusion protein is detected and/or manipulated using the label in a system designed for recognizing and/or handling the label. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching mols. to the fusion proteins that introduce a new phys. or chem. property to the fusion protein. Examples of such mols. are, among others, spectroscopic probes or radioactively labeled mols., affinity tags, mols. generating reactive or hydroxyl radicals, cross-linkers and lipid. AGT in fusion protein is a human variant wherein Asn157 is replaced by Gly and Ser159 by Glu, and the one wherein Gly160 is replaced by Ala or Trp. AGT in fusion protein is a human variant wherein Asn157 is replaced by Ser, Ser159 by His, and Gly160 by Asn. [on SciFinder (R)]

(Ecole Polytechnique Federale de Lausanne (EPFL), Switz.).2004

Chargement

A method for labeling acyl carrier protein (ACP) fusion proteins using derivs. of CoA is described. The method relies on the transfer of a label from a CoA type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homolog thereof. The method allows detecting and manipulating the fusion protein, both in vitro and in vivo, by attaching mols. to the fusion proteins that introduce a new phys. or chem. property to the fusion protein. Examples of such labels are, among others, spectroscopic probes or reporter mols., affinity tags, mols. generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or mols. suitable for the immobilization of the fusion protein. Synthesis of a series of reporter mol. conjugates, including digoxigenin, Cy3 and Cy5, with CoA is reported. [on SciFinder (R)]

(EPFL-Ecole Polytechnique Federale de Lausanne, Switz.).2004

Chargement