Protein engineering using directed evolution

Background

Protein engineering was 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 showing 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) and UDP-Glucuronosyltransferases (UGTs) are liver enzymes that detoxify a variety of substrates by transferring sulfate and glucuronic acid, respectively, to a variety of acceptor molecules bearing a hydroxyl or an amine group. Sulfation or glucuronidation renders the product more readily excretable or less pharmacologically active. The diversity of acceptor compounds for cytosolic SULTs and UGTs 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. They are also of significant biotechnological importance, as they could provide a means for detoxification of a wide variety of xenobiotics that possess steroid-like activity and are increasingly found in many drinking water systems. Using directed evolution, we aim to improve the detoxification properties of SULTs and GUTs 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. Lysosomal storage diseases are caused by genetic defects in lysosomal enzymes which are responsible for the degradation of glycopepetides and glycolipids in the lysosome. The most prevalent lysosomal storage disease, Gaucher’s disease, is caused by the inefficient folding and trafficking of certain variants of β-glucocerebrosidase (β-glu). Recently, it was shown that many of the mutations leading to Gaucher’s disease interfere with the folding of β-Glu in the ER and/or trafficking to the lysosome. Current treatment of Gaucher's disease includes replacement of the defective enzyme with recombinant enzyme infusion or inhibition of glucosylceramide production. We plan to develop a novel approach based on peptide engineering for stabilization of mutant enzymes. The engineered peptides will bind the mutated enzyme and stabilize the active enzyme form in the ER, thus preventing its degradation. This approach may lead to enzyme stabilization at different peptide binding sites, thereby allowing stabilization of different mutations that compromise enzyme folding and stability.

Selected publications

Aharoni A., Gaidukov L., Khersonsky O., Gould S.M., Roodveldt C., Tawfik D.S. (2005) The ‘evolvability’ of promiscuous protein functions. Nature Genetics, 37:73-76.

Aharoni A., Griffiths A. D., Tawfik D.S. (2005) High-throughput screens and selections of enzyme encoding genes. Curr. Opin. Chem. Biol., 9:210-216.

Aharoni A., Thieme K., Chiu C.P.C., Buchini S., Lairson L.L., Chen H., Strynadka N.C.J., Wakarchuk W.W., Withers S.G. (2006) Novel high throughput screening methodology for the directed evolution of glycosyltransferases. Nature Methods, 3:609-614.