Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases

• Published in: ACS chemical biology Vol. 2; no. 5; pp. 337 - 346
• Main Authors: Zhou, Zhe, Cironi, Pablo, Lin, Alison J, Xu, Yangqing, Hrvatin, Siniša, Golan, David E, Silver, Pamela A, Walsh, Christopher T, Yin, Jun
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.05.2007
• Subjects: 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
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb700054k

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, k cat/K m of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the k cat/K m 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.