Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases.
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.
Zhou
Zhe
Z
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Cironi
Pablo
P
Lin
Alison J
AJ
Xu
Yangqing
Y
Hrvatin
Sinisa
S
Golan
David E
DE
Silver
Pamela A
PA
Walsh
Christopher T
CT
Yin
Jun
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D019151:Peptide Library; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015335:Molecular Probes; D010446:Peptide Fragments; D019151:Peptide Library; D011956:Receptors, Cell Surface; C082662:phosphopantetheinyl transferase; C073125:acpS protein, E coli; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase