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

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.

A general method for the covalent labeling of fusion proteins with small molecules in vivo

Thomas Gronemeyer, Kai Johnsson, Antje Keppler, Horst Pick, Horst Vogel

Characterizing the movement, interactions, and chem. microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic mols. capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small mol. to a protein, the formation of stable complexes between biarsenical compds. and peptides contg. cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

2003

Method for identification of fragmentation sites in a reporter protein in the development of split protein sensors

Kai Johnsson, Petra Tafelmeyer

A combinatorial method for the generation of new split-protein sensors that identifies sites at which a functional domain can be split to give two moieties that can be reconstituted to give a reporter or selectable activity is described. The method is demonstrated with the gene Trp1 N-(5'-phosphoribosyl)-anthranilate isomerase from Saccharomyces cerevisiae, which is (b/a)8-barrel enzyme. The resulting protein is described as a split-Trp sensor. 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. Selection for tryptophan prototrophy is rapid and effective and can be used for high-throughput interaction screening. A series of deletion derivs. of the region of the gene encoding the catalytic domain of the protein was constructed by DNase I digestion of restriction fragments. These were combined with sequences encoding leucine zippers and tested pairwise for their ability to lift a tryptophan auxotrophy in yeast. Use of the split-Trp protein sensor to identify the interaction of the SEC62 and SEC63 gene products in the cell membrane of S. cerevisiae is demonstrated. [on SciFinder (R)]

(Ecole Polytechnique Federale De Lausanne, Switz.).2005

O6-alkylguanine-DNA alkyltransferase as a new tool for functional proteomics

Anke Arnold

Recently the sequencing project of the human genome has been completed. The interest of the post-sequencing-era is shifting nowadays towards the investigation of the biological function and localization of the encoded proteins. Proteins are essential parts of all living organisms and participate in every process in the living cell. Despite their importance, the detailed function of the majority of all proteins remains still unknown. Therefore high-throughput methods are needed for the systematic examination of proteins. The first part of this thesis describes the development of a novel functional protein microarray, taking advantage of the highly selective reaction of the human O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. By functionalization of otherwise bioinert glass surfaces with the substrate O6-benzylguanine, AGT fusion proteins can be selectively and covalently immobilized on a solid surface (Figure S1). Due to the high selectivity of the covalent immobilization the main advantage of this AGT based approach is that fusion proteins can be immobilized directly out of cell extracts without prior purification. By analyzing a set of literature known protein-protein interactions, protein-small molecule interactions and posttranslational modifications we demonstrate the feasibility of this approach (Chapter 3). Figure S1: Principle of an AGT-based protein microarray in which x can be any protein of interest Furthermore, the development of surface materials suitable for functional protein microarrays is described. The generated surfaces allow immobilization of proteins in defined orientation and density. Intrinsically bioinert polymer brushes were generated in collaboration with the Laboratory of Polymers at the EPFL. For the production of these bioinert brush-surfaces, surface-initiated atom transfer radical polymerization (SI-ATRP) was used (Chapter 4). Additionally we addressed the importance of the orientation of immobilized proteins on solid surfaces. The immobilization of fusion proteins with random orientation on aldehyde-coated glass slides was compared with immobilization in a defined orientation on O6-benzylguanine functionalyzed polymer brushes. Subsequently, retention of activity of the proteins upon immobilization on the two different surfaces was analyzed. We demonstrated that a defined orientation of immobilized proteins is advantageous over random immobilization due to the fact that proteins, immobilized using the AGT approach, are attached in a defined orientation with their active site facing the solution phase, whereas random immobilization of proteins can result in hidden active sites (Chapter 5). In the second part of this thesis the problem of insolubility of recombinant expressed proteins is addressed. We analyzed the protein expression of a set of proteins, which were prior determined to be insoluble when expressed in E. coli. The proteins were expressed as AGT fusion proteins either in E. coli or M. smegmatis, with the goal to identify a suitable host for soluble protein expression with high yield. The AGT approach was exploited to screen for expression of soluble proteins and for the determination of the expression yields. We showed that fusion to AGT does not influence the solubility of the expressed proteins and that the expression yield in E. coli does not significantly differ from the yield in M. smegmatis, where proteins were expressed with less degradation. However, we assessed E. coli. to be the more suitable host for multi-parallel protein expression due to its easy handling and fast growth (Chapter 6). The gained knowledge of this project was transferred to the investigation of proteins from Mycobacterium tuberculosis, which is presented in the last part of this thesis. A better understanding of M. tuberculosis, with the goal of unveiling and validating new therapeutic targets, is an imperative need to improve the control and treatment of tuberculosis disease. We focused in particular on the bacterial two component adaptation systems (TCS). Since these systems do not exist in mammals they present attractive candidates for new drug targets. In order to gain a better understanding of these adaptation systems we focused on one specific TCS, SenX/RegX, one of the first identified and well established TCS, which is believed to be implicated in virulence. A radioactive in vitro phosphorylation assay was developed, which allowed the identification of two new in vitro substrates for histidine kinase SenX, namely DevR and NarL. These results confirm the hypothesis that TCS might not only consist of two components, but that cross-talk between different TCS exist in order to adapt rapidly to environmental changes.

EPFL2007

O6-alkylguanine-DNA alkyltransferase as a new tool for functional proteomics

Anke Arnold

EPFL2007