Publication

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

Petra Tafelmeyer
2004
EPFL thesis
Abstract

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.

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Protein–protein interaction
Protein–protein interactions (PPIs) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces, hydrogen bonding and the hydrophobic effect. Many are physical contacts with molecular associations between chains that occur in a cell or in a living organism in a specific biomolecular context. Proteins rarely act alone as their functions tend to be regulated.
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Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.
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