Protein function microarrays based on self-immobilizing and self-labeling fusion proteins

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

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

Development of a new reporter system for the detection of protein-protein interactions in living cells

Petra Tafelmeyer

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. In general, two interacting proteins are expressed as fusion proteins with a pair of inactive fragments of a reporter enzyme. Interaction-induced reassembly of the two fragments then results in a functional enzyme and a detectable phenotypic readout. Despite the constantly expanding repertoire of methods, new split-protein sensors that could detect and screen for protein-protein interactions both in the cytosol and in the membrane would be very useful. In a first attempt to create new split-protein sensors, cytochrome c peroxidase (CCP) from the yeast Saccharomyces cerevisiae was rationally dissected into two fragments, which were fused to two interacting proteins. Activity of reassembled peroxidase might be visually detected using a simple colony screen [1]. However, this approach failed due to the insolubility of the chosen fragments. A random approach based on circular permutation originally developed by Graf and Schachmann was therefore adapted to isolate suitable fragmentation sites [2]. Unfortunately, only split-proteins expressing quasi wild-type protein were isolated, resulting from cuts close to the N or the C terminus of CCP. Two reasons can account for this: (i) the fragile active site environment of CCP could considerably hamper the fragmentation of the enzyme; (ii) the method itself favors the isolation of quasi wild-type proteins. The combinatorial method for the generation of new split-protein sensors was therefore further modified to circumvent the isolation of quasi wild-type proteins and successfully applied to the (β/α)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. 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. In addition, split-Trp can be used as reporter for the detection of small molecule-protein interactions. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening. Furthermore, the combinatorial approach should be able to generate split-protein sensors of almost any protein, thereby yielding tailor-made sensors for different applications.

EPFL2004

Methods using O6-alkylguanine-DNA alkyltransferases

Kai Johnsson, Antje Keppler

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)]

(Ecole Polytechnique Federale De Lausanne, Switz.).2002

Development of a new reporter system for the detection of protein-protein interactions in living cells

Petra Tafelmeyer

EPFL2004