Abstract
Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.
MeSH terms
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Aminoacyltransferases / genetics*
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Aminoacyltransferases / metabolism
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Calcium / metabolism*
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Cell Membrane / chemistry
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Cysteine Endopeptidases / genetics*
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Cysteine Endopeptidases / metabolism
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Mutation
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Peptidyl Transferases / genetics*
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Peptidyl Transferases / metabolism
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Staining and Labeling / methods*
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Staphylococcus aureus / enzymology
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Staphylococcus aureus / genetics*
Substances
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Bacterial Proteins
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Aminoacyltransferases
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sortase A
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Peptidyl Transferases
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Cysteine Endopeptidases
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Calcium
Grants and funding
HJJ was funded by a fellowship from the Dana-Farber Center for Cancer Immunology Research. GCA was funded by the Harvard PRISE program. SKD and JRI were funded by the Claudia Adams Barr Foundation. SKD was funded by the Dana-Farber Cancer Institute/Novartis Drug Discovery Program.