Abstract: The invention provides a method of crosslinking two objects of interest, comprising the steps of: i) providing a fusion protein comprising at least a first protein and a second protein, wherein both the first and the second protein are, based on their structure and function, capable of forming a covalent bond with given substrates, and which first and second proteins are of substantially non-overlapping substrate selectivity, preferably of different substrate specificity; ii) providing a first object of interest, comprising a substrate moiety for the first protein of the said fusion protein, and providing a second object of interest, comprising a substrate moiety for the second protein of the said fusion protein; and iii) reacting said first protein of the fusion protein with the substrate moiety of said first object, and reacting said second protein of the fusion protein with the substrate moiety of said second object, thereby covalently crosslinking the first object to the second object via the said fusion

Abstract: A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating 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 labels 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 immobilization of the fusion protein.

Abstract: The invention concerns a combinatorial method for the generation of new split-protein sensors, and its application towards the (?/?)8-barrel enzyme N-(5?-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae is demonstrated. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

Abstract: The invention relates to substrates for O6-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X—CH2—R3—R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group —R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate, or a further group —R3 CH2—X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.

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 06-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted 06-alkylguanine substrates.

Abstract: The invention concerns a combinatorial method for the generation of new split-protein sensors, and its application towards the (?/?a)8-barrel enzyme N-(5?-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae is demonstrated. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

Abstract: A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating 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 labels 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 immobilization of the fusion protein.

Abstract: 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 labelled 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 recognising and/or handling the label.

Abstract: The present invention relates to methods of transferring a label from novel substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and O6-alkylguanine-DNA alkyltransferase fusion proteins, and to novel substrates suitable in such methods. Proteins of interest are incorporated into an AGT fusion protein, the AGT fusion protein is contacted with particular AGT substrates carrying a label, and the AGT fusion protein is detected and optionally further manipulated using the label in a system designed for recognising and/or handling the label. The particular AGT substrates used in the method of the invention are O6-substituted guanine derivatives or related nitrogen containing hydroxy-heterocycles and their sulfur analogs wherein the O6-substituent is an activated methyl derivative suitable for transfer from guanine or the corresponding heterocycle to AGT, and further carrying a label or a plurality of same or different labels.

Abstract: 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 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.