Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases

ACS Chem Biol. 2007 May 22;2(5):337-46. doi: 10.1021/cb700054k. Epub 2007 Apr 27.


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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus subtilis / enzymology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Catalysis
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Molecular Probes / chemistry
  • Molecular Probes / genetics*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics*
  • Peptide Library
  • Protein Processing, Post-Translational
  • Receptors, Cell Surface / chemistry*
  • Substrate Specificity
  • Transferases (Other Substituted Phosphate Groups) / chemistry*
  • Transferases (Other Substituted Phosphate Groups) / metabolism
  • Transferases / chemistry*
  • Transferases / metabolism


  • Bacterial Proteins
  • Escherichia coli Proteins
  • Molecular Probes
  • Peptide Fragments
  • Peptide Library
  • Receptors, Cell Surface
  • phosphopantetheinyl transferase
  • acpS protein, E coli
  • Transferases
  • Transferases (Other Substituted Phosphate Groups)
  • holo-(acyl-carrier-protein) synthase