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An in vitro compartmentalization‐based method for the selection of bond‐forming enzymes from large libraries
Author(s) -
Gianella Paul,
Snapp Erik L.,
Levy Matthew
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25939
Subject(s) - compartmentalization (fire protection) , sortase a , intracellular , enzyme , nucleic acid , biochemistry , in vitro , cytoplasm , mutant , biology , directed evolution , computational biology , chemistry , microbiology and biotechnology , bacterial protein , gene
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 (10 6 –10 7 ), 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 10 12 protein variants by allowing beads to be overloaded with up to 10 4 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. © 2016 Wiley Periodicals, Inc.