An in vitro compartmentalization-based method for the selection of bond-forming enzymes from large libraries

We have developed a generalized in vitro compartmentalization-based bead display selection strategy that allows for the identification of enzymes that can perform ligation reactions. Although a number of methods have been developed to evolve such enzymes, many of them are limited in library size (10⁶–10⁷), do not select for enzymes using a scheme that allows for multiple turnover, or only work on enzymes specific to nucleic acids. This approach is amenable to screening libraries of up to 10¹² protein variants by allowing beads to be overloaded with up to 10⁴ unique mutants. Using this approach we isolated a variant of sortase A from Staphylococcus aureus that shows a 114-fold enhancement in k_cat/K_M in the absence of calcium compared to the wild-type and improved resistance to the inhibitory effects of cell lysates. Unlike the wild-type protein, the newly selected variant shows intracellular activity in the cytoplasm of eukaryotic cells where it may prove useful for intracellular labeling or synthetic biological applications. Biotechnol. Bioeng. 2016;113: 1647–1657.