Abstract
Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacillus subtilis / enzymology
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Bacterial Proteins / chemistry*
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Bacterial Proteins / metabolism
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Catalysis
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Cloning, Molecular
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli / enzymology
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / metabolism
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HeLa Cells
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Humans
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Models, Molecular
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Molecular Probes / chemistry
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Molecular Probes / genetics*
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Molecular Sequence Data
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Peptide Fragments / chemistry
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Peptide Fragments / genetics*
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Peptide Library
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Protein Processing, Post-Translational
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Receptors, Cell Surface / chemistry*
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Substrate Specificity
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Transferases (Other Substituted Phosphate Groups) / chemistry*
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Transferases (Other Substituted Phosphate Groups) / metabolism
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Transferases / chemistry*
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Transferases / metabolism
Substances
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Bacterial Proteins
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Escherichia coli Proteins
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Molecular Probes
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Peptide Fragments
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Peptide Library
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Receptors, Cell Surface
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phosphopantetheinyl transferase
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acpS protein, E coli
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Transferases
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Transferases (Other Substituted Phosphate Groups)
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holo-(acyl-carrier-protein) synthase