Evolution of bond-forming enzymes
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Irwin Chen, Brent M. Dorr
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Abstract
Strategies, systems, methods, reagents, and kits for the directed evolution of bond-forming enzymes are provided herein. Evolution products, for example, evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are also provided herein, as are methods for using such evolved bond-forming enzymes. Kits comprising materials, reagents, and cells for carrying out the directed evolution methods described herein are also provided.
Claims32
A sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of Staphylococcus aureus (S. aureus) Sortase A as provided as SEQ ID NO:
- 1 or to amino acid residues 1-150 of SEQ ID NO:
- 21, wherein the amino acid sequence of the sortase comprises two or more mutations selected from the group consisting of P94S, P94R, E106G, F122Y, K154R, D160N, D165A, G174S, K190E, and K196T.
A sortase catalyzing transpeptidation of substrates other than LPETG (SEQ ID NO:
- 32), the sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO:
- 1 or to amino acid residues 1-150 of SEQ ID NO:
- 21,
- wherein the amino acid sequence comprises two or more mutations selected from the group consisting of P86L, P94R, N98S, A104T, A118T, F122S, D124G, N127S, K134R, D160N, D165A K173E, K177E, K190E, and K196T.
Dependencies are inferred from the claim text.