Available Technology
Directed evolution of bond-forming enzymes using yeast display: application to sortase A
Technology:
Directed evolution method
Markets Addressed
A method to routinely generate custom-designed enzymes that efficiently catalyze any desired bond-forming reaction is a long-standing goal of the molecular life sciences. While current methods for the directed evolution of enzymes have resulted in some remarkable successes, they generally suffer from limitations in reaction scope. Directed evolution strategies that are general for any bond-forming reaction would complement current methods that rely on screenable reactions or selectable properties of the substrate or product.
Innovations and Advantages
Researchers in the laboratory of Professor David Liu have developed a general strategy for the evolution of proteins that catalyze bond-forming (coupling) reactions. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher.
The system was successfully used to evolve sortase A for improved catalytic transpeptidation activity. After just eight rounds of screening, the researchers isolated variants of sortase A with up to a 140-fold increase in coupling activity compared with the starting wild-type enzyme. The evolved sortase variants were also shown to enable the much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase.
Because this method does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods. Beyond improving existing activities of natural proteins for research, industrial, and medicinal use, this enzyme evolution strategy will be valuable in the engineering of artificial proteins with new, tailor-made catalytic activities.
A general strategy for the evolution of bond-forming catalysts using yeast display.
Yeast cells display the enzyme library extracellularly as a fusion to the Aga2p cell surface mating factor, which is covalently bound to the Aga1p mating factor with a reactive handle (S6 peptide) that enables covalent attachment of substrate A to cells using Sfp phosphopantetheinyl transferase. Substrate B linked to an affinity handle (e.g., biotin, represented by the gray circle) is added to the substrate A–conjugated yeast display enzyme library. Active library members will predominantly catalyze the pseudointramolecular A─B bond formation between affinity handle-linked substrate B and substrate A molecules on their own host cell.
Additional Information
Intellectual Property Status: Patent(s) pending
Publications:
A general strategy for the evolution of bond-forming enzymes using yeast display, Chen, I; Brent, MD; Liu, DR. Proc. Natl. Acad. Sci. USA 108 (28), 11399-11404 (2011), published online 22 June 2011; doi:10.1073/pnas.1101046108
Related technology:
Evolved sortase A variants with improved catalytic activity
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Inventor(s):
Chen, Irwin
Dorr, Brent
Liu, David R.
Categories:
For further information, please contact:
Vivian Berlin, Director of Business Development
(617) 496-0474
Reference Harvard Case #4197