EVOLUTION OF BOND-FORMING ENZYMES

摘要

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.

主权项

1. A method for directed evolution of bond-forming enzymes, the method comprising
(a) providing a cell population in which
(i) a first cell surface protein or a cell in the cell population is conjugated to a candidate bond-forming enzyme, wherein different cells within the population of cells comprise different candidate bond-forming enzymes conjugated to the cell surface protein;(ii) a second cell surface protein is conjugated to a substrate A; (b) contacting the cell population with a substrate B conjugated to a detectable label under conditions suitable for the bond-forming enzyme to form a bond between substrate A and substrate B; and (c) identifying and/or isolating a cell that is conjugated to substrate B.