Ph.D.: Weizmann Institute of Science, Israel
Post-doctorate: Weizmann Institute of Science, Israel and
University of British Columbia, Canada
Position: Senior Lecturer
Department of Life Sciences
Faculty of Natural Sciences
E-mail: aaharoni@bgu.ac.il
Webpage:http://www.bgu.ac.il/~aaharoni
Protein engineering using directed evolution
Protein engineering has been used extensively in the past twenty years for the study of enzyme structure-function and evolution. Recently, protein engineering using directed evolution has proven to be highly successful, yielding proteins demonstrating increased stability under extreme conditions, increased solubility for expression in heterologous systems, and proteins with novel reaction and substrate specificities. Directed evolution implements an iterative Darwinian optimization process, whereby the fittest variants are selected from a collection of random mutations. Improved variants are identified and isolated by screening or selection for the property of interest. This approach is particularly advantageous in cases in which no prior knowledge of a protein’s mechanism and structure is available.
Current research
1. Cytosolic sulfotransferases (SULTs) are liver enzymes that detoxify a variety of substrates by transferring sulfate to a variety of acceptor molecules bearing a hydroxyl or an amine group. Sulfation renders the product more readily excretable or less pharmacologically active. The diversity of acceptor compounds for cytosolic SULTs is remarkable, ranging in size, shape and flexibility, from ethanol to steroids. These enzymes play important roles in a variety of biological functions, such as modulating the levels of hormones and neurotransmitters. Using directed evolution, we aim to improve the detoxification properties of SULTs by implementing a new high throughput screening methodology that allows for the screening of millions of mutant enzymes in parallel for increases in catalytic efficiency. Improved mutant enzymes may find ex-vivo biotechnological applications, such as in bioremediation.
2. DNA replication and gene transcription are two ubiquitous biological processes in all living organisms. We are currently developing new tools to study these processes using directed evolution methodology. We are focusing on the proliferating cellular nuclear antigen (PCNA) which is a hub protein orchestrating the DNA replication process in eukaryotic cells. We aim to elucidate the importance of PCNA-protein interaction for DNA replication, repair and cell viability. Another project is focused on the study of stress-related transcription factor (TF) in yeast. We aim to generate TF mutants that increase the ability of yeast to survive under different stress conditions in order to study the molecular basis for higher tolerance to different conditions and the linkage between the yeast responses to different stress conditions.
3. Engineering of therapeutic proteins for increased stability and binding affinity – We are focusing in generating improved soluble receptors for inhibiting pro-inflammatory cytokines involved in a variety of different autoimmune diseases. Our initial efforts were focused on the development of a unique, soluble IL-17 receptor therapeutic with improved properties, that was shown e to inhibit psoriasis plaque formation in a mouse model. This protein constitutes an important drug candidate for the treatment of psoriasis in humans.
Selected publications
Fridman Y., Palgi N., Dovrat D., Ben-Aroya S., Hieter P., Aharoni A. (2010). Subtle Alterations in PCNA-partner Interactions Severely Impair DNA Replication and Repair. PLoS Biology 8(10):e100.
Sadeh A., Baran D., Volokh M., Aharoni A. (2012). Conserved Motifs in the Msn2-Activating Domain are Important for Msn2-mediated Yeast Stress Response. J. Cell. Sci. 125:3333-3342.
Zamir L., Zaretsky M., Fridman Y., Ner-Gaon H., Rubin E., Aharoni A. (2012). Tight co-evolution of PCNA-partner interaction networks in fungi leads to inter-species network incompatibility. Proc. Natl. Acad. Sci. U.S.A 109(7):E406-414.
Amar D., Berger I., Amara N., Tafa G., Meijler M., Aharoni A. (2012). The Transition of Human Estrogen Sulfotransferase from Generalist to Specialist using Directed Enzyme Evolution. J. Mol. Biol. 416(1):21-32.
Zaretsky M., Etzyoni R., Kaye K., Sklair-Tavron L., Aharoni A. (2013). Directed Evolution of a Soluble Human IL-17A Receptor for the Inhibition of Psoriasis Plaque in Mice Model. Chemistry and Biology 20:202-211.
