title: An in vitro compartmentalization-based method for the selection of bond-forming enzymes from large libraries.
abstract: 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 kcat /KM 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1301 Morris Park Ave, Bronx, New York, 10461.
authors.forename: Paul
authors.initials: P
authors.lastname: Gianella
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Erik L
authors.initials: EL
authors.lastname: Snapp
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1301 Morris Park Ave, Bronx, New York, 10461. matthew.levy@einstein.yu.edu.|Price Center for Genetics and Translational Medicine, 1301 Morris Park Ave, Bronx, New York, 10461. matthew.levy@einstein.yu.edu.
authors.forename: Matthew
authors.initials: M
authors.lastname: Levy
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012097:Repressor Proteins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D019151:Peptide Library; D012097:Repressor Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: FACS; biotin protein ligase (BirA); directed evolution; in vitro compartmentalization; microbead display; sortase A