The PA Tag: A Versatile Peptide Tagging System in the Era of Integrative Structural Biology

  • Zuben P. Brown
  • Junichi TakagiEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1105)


We have recently developed a novel protein tagging system based on the high affinity interaction between an antibody NZ-1 and its antigen PA peptide, a dodecapeptide that forms a β-turn in the binding pocket of NZ-1. This unique conformation allows for the PA peptide to be inserted into turn-forming loops within a folded protein domain and the system has been variously used in general applications including protein purification, Western blotting and flow cytometry, or in more specialized applications such as reporting protein conformational change, and identifying subunits of macromolecular complexes with electron microscopy. Thus the small and “portable” nature of the PA tag system offers a versatile and powerful tool that can be implemented in various aspects of integrative structural biology.


Protein tagging Affinity purification Monoclonal antibody Peptide insertion EM label 


  1. Arimori T, Kitago Y, Umitsu M et al (2017) Fv-clasp: an artificially designed small antibody fragment with improved production compatibility, stability, and crystallizability. Structure 25:1611–1622CrossRefGoogle Scholar
  2. Boisset N, Radermacher M, Grassucci R et al (1993) Three-dimensional immunoelectron microscopy of scorpion hemocyanin labeled with a monoclonal fab fragment. J Struct Biol 111:234–244CrossRefGoogle Scholar
  3. Boisset N, Penczek P, Taveau JC et al (1995) Three-dimensional reconstruction of androctonus australis hemocyanin labeled with a monoclonal fab fragment. J Struct Biol 115:16–29CrossRefGoogle Scholar
  4. Bonasio R, Carman CV, Kim E et al (2007) Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots. Proc Natl Acad Sci U S A 104:14753–14758CrossRefGoogle Scholar
  5. Brown Z, Arimori T, Iwasaki K et al (2017) Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy. J Struct Biol 201:247–251CrossRefGoogle Scholar
  6. Buchel C, Morris E, Orlova E et al (2001) Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis. J Mol Biol 312:371–379CrossRefGoogle Scholar
  7. Bui KH, Sakakibara H, Movassagh T et al (2008) Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella. J Cell Biol 183:923–932CrossRefGoogle Scholar
  8. Calleja V, Ameer-Beg SM, Vojnovic B et al (2003) Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem J 372:33–40CrossRefGoogle Scholar
  9. Chen J, Sawyer N, Regan L (2013) Protein-protein interactions: general trends in the relationship between binding affinity and interfacial buried surface area. Protein Sci 22:510–515CrossRefGoogle Scholar
  10. Ciferri C, Lander GC, Maiolica A et al (2012) Molecular architecture of human polycomb repressive complex 2. elife 1:e00005CrossRefGoogle Scholar
  11. Ciferri C, Lander GC, Nogales E (2015) Protein domain mapping by internal labeling and single particle electron microscopy. J Struct Biol 192:159–162CrossRefGoogle Scholar
  12. Dennison SM, Anasti KM, Jaeger FH et al (2014) Vaccine-induced HIV-1 envelope gp120 constant region 1-specific antibodies expose a CD4-inducible epitope and block the interaction of HIV-1 gp140 with galactosylceramide. J Virol 88:9406–9417CrossRefGoogle Scholar
  13. Dinculescu A, McDowell JH, Amici SA et al (2002) Insertional mutagenesis and immunochemical analysis of visual arrestin interaction with rhodopsin. J Biol Chem 277:11703–11708CrossRefGoogle Scholar
  14. Dyson HJ, Lerner RA, Wright PE (1988) The physical basis for induction of protein-reactive antipeptide antibodies. Annu Rev Biophys Biophys Chem 17:305–324CrossRefGoogle Scholar
  15. Evan GI, Lewis GK, Ramsay GB, J. M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616CrossRefGoogle Scholar
  16. Facey SJ, Kuhn A (2003) The sensor protein KdpD inserts into the Escherichia coli membrane independent of the sec translocase and YidC. Eur J Biochem 270:1724–1734CrossRefGoogle Scholar
  17. Field J, Nikawa J, Broek D et al (1988) Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8: 2159–2165CrossRefGoogle Scholar
  18. Flemming D, Thierbach K, Stelter P et al (2010) Precise mapping of subunits in multiprotein complexes by a versatile electron microscopy label. Nat Struct Mol Biol 17:775–778CrossRefGoogle Scholar
  19. Forsberg BO, Aibara S, Kimanius D et al (2017) Cryo-EM reconstruction of the chlororibosome to 3.2 Å resolution within 24 h. IUCr J 4:723–727CrossRefGoogle Scholar
  20. Fujii Y, Kaneko M, Neyazaki M et al (2014) PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expres Purif 95:240–247CrossRefGoogle Scholar
  21. Fujii Y, Matsunaga Y, Arimori T, et al. (2016a) Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state. J. Cell Sci., 1512-1522CrossRefGoogle Scholar
  22. Fujii Y, Kaneko MK, Kato Y (2016b) MAP tag: a novel tagging system for protein purification and detection. Monoclon Antib Immunodiagn Immunother 35:293–299CrossRefGoogle Scholar
  23. Fujii Y, Kaneko MK, Ogasawara S et al (2017) Development of RAP tag, a novel tagging system for protein detection and purification. Monoclon Antib Immunodiagn Immunother 36:68–71CrossRefGoogle Scholar
  24. Guruprasad K, Rajkumar S (2000) Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials. J Biosci 25:143–156CrossRefGoogle Scholar
  25. Hancock DC, O’Reilly NJ (2005) Synthetic peptides as antigens for antibody production. Methods Mol Biol 295:13–26PubMedGoogle Scholar
  26. Heuser T, Raytchev M, Krell J et al (2009) The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. J Cell Biol 187:921–933CrossRefGoogle Scholar
  27. Heuser T, Barber CF, Lin J et al (2012) Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein. Proc Natl Acad Sci U S A 109:E2067–E2076CrossRefGoogle Scholar
  28. Hirai H, Yasui N, Yamashita K et al (2017) Structural basis for ligand capture and release by the endocytic receptor ApoER2. EMBO Rep 18:982–999CrossRefGoogle Scholar
  29. Hopp TP, Prickett KS, Price VL et al (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat Biotechnol 6:1204–1210CrossRefGoogle Scholar
  30. Humphries MJ, Symonds EJ, Mould AP (2003) Mapping functional residues onto integrin crystal structures. Curr Opin Struct Biol 13:236–243CrossRefGoogle Scholar
  31. Irannejad R, Tomshine JC, Tomshine JR et al (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–538CrossRefGoogle Scholar
  32. Kato Y, Kaneko MK, Kuno A et al (2006) Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain. Biochem Biophys Res Commun 349:1301–1307CrossRefGoogle Scholar
  33. Kato K, Nishimasu H, Okudaira S et al (2012) Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling. Proc Natl Acad Sci U S A 109:16876–16881CrossRefGoogle Scholar
  34. Kelly DF, Lake RJ, Middelkoop TC et al (2010) Molecular structure and dimeric organization of the notch extracellular domain as revealed by electron microscopy. PLoS One 5:e10532CrossRefGoogle Scholar
  35. Kendall RT, Senogles SE (2006) Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution. Neurosci Lett 393:155–159CrossRefGoogle Scholar
  36. Kitago Y, Nagae M, Nakata Z et al (2015) Structural basis for amyloidogenic peptide recognition by sorLA. Nat Struct Mol Biol 22:199–206CrossRefGoogle Scholar
  37. Koide S (2009) Engineering of recombinant crystallization chaperones. Curr Opin Struct Biol 19:449–457CrossRefGoogle Scholar
  38. Maina CV, Riggs PD, Grandea AG 3rd et al (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74:365–373CrossRefGoogle Scholar
  39. Matoba K, Mihara E, Tamura-Kawakami K et al (2017) Conformational freedom of the lrp6 ectodomain is regulated by n-glycosylation and the binding of the wnt antagonist dkk1. Cell Rep 18:32–40CrossRefGoogle Scholar
  40. Matsunaga Y, Bashiruddin NK, Kitago Y et al (2016) Allosteric inhibition of a semaphorin 4d receptor plexin b1 by a high-affinity macrocyclic peptide. Cell Chem Biol 23:1341–1350CrossRefGoogle Scholar
  41. Mercogliano CP, Derosier DJ (2007) Concatenated metallothionein as a clonable gold label for electron microscopy. J Struct Biol 160:70–82CrossRefGoogle Scholar
  42. Morita J, Kano K, Kato K et al (2016) Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase. Sci Rep 6:20995CrossRefGoogle Scholar
  43. Morlacchi S, Sciandra F, Bigotti MG et al (2012) Insertion of a myc-tag within alpha-dystroglycan domains improves its biochemical and microscopic detection. BMC Biochem 13:14CrossRefGoogle Scholar
  44. Nagae M, Nishikawa K, Yasui N et al (2008) Structure of the F-spondin reeler domain reveals a unique beta-sandwich fold with a deformable disulfide-bonded loop. Acta Crystallogr D Biol Crystallogr 64:1138–1145CrossRefGoogle Scholar
  45. Nishimasu H, Okudaira S, Hama K et al (2011) Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. Nat Struct Mol Biol 18:205–212CrossRefGoogle Scholar
  46. Nishino Y, Yasunaga T, Miyazawa A (2007) A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy. J Electron Microsc 56:93–101CrossRefGoogle Scholar
  47. Nogi T, Sangawa T, Tabata S et al (2008) Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification. Protein Sci 17: 2120–2126CrossRefGoogle Scholar
  48. Nogi T, Yasui N, Mihara E et al (2010) Structural basis for semaphorin signalling through the plexin receptor. Nature 467:1123–1127CrossRefGoogle Scholar
  49. Pigino G, Bui KH, Maheshwari A et al (2011) Cryoelectron tomography of radial spokes in cilia and flagella. J Cell Biol 195:673–687CrossRefGoogle Scholar
  50. Prasad BV, Burns JW, Marietta E et al (1990) Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy. Nature 343:476–479CrossRefGoogle Scholar
  51. Sangawa T, Tabata S, Suzuki K et al (2013) A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein. Protein Sci 22:840–850CrossRefGoogle Scholar
  52. Sassenfeld HM, Brewer SJ (1984) A polypeptide fusion designed for the purification of recombinant proteins. Bio-Technol 2:76–81Google Scholar
  53. Schmidt TG, Skerra A (2007) The strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535CrossRefGoogle Scholar
  54. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia-coli as fusions with glutathione s-transferase. Gene 67:31–40CrossRefGoogle Scholar
  55. Stofkohahn RE, Carr DW, Scott JD (1992) A single step purification for recombinant proteins – characterization of a microtubule associated protein (map-2) fragment which associates with the type-II camp-dependent protein-kinase. FEBS Lett 302:274–278CrossRefGoogle Scholar
  56. Tabata S, Nampo M, Mihara E et al (2010) A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system. J Proteome 73:1777–1785CrossRefGoogle Scholar
  57. Takagi J, Springer TA (2002) Integrin activation and structural rearrangement. Immunol Rev 186:141–163CrossRefGoogle Scholar
  58. Takagi J, Petre BM, Walz T et al (2002) Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110:599–611CrossRefGoogle Scholar
  59. Takeda H, Zhou W, Kido K et al (2017) CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag. PLoS One 12:e0178246CrossRefGoogle Scholar
  60. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533CrossRefGoogle Scholar
  61. Walker F, Orchard SG, Jorissen RN et al (2004) CR1/CR2 interactions modulate the functions of the cell surface epidermal growth factor receptor. J Biol Chem 279:22387–22398CrossRefGoogle Scholar
  62. Wang H, Han W, Takagi J et al (2018) Yeast inner-subunit PA-NZ-1 labeling strategy for accurate subunit identification in a macromolecular complex through cryo-EM analysis. J Mol Biol 430:1417–1425CrossRefGoogle Scholar
  63. Yano T, Takeda H, Uematsu A et al (2016) AGIA tag system based on a high affinity rabbit monoclonal antibody against human dopamine receptor D1 for protein analysis. PLoS One 11:e0156716CrossRefGoogle Scholar
  64. Zhu J, Luo BH, Xiao T et al (2008) Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol Cell 32:849–861CrossRefGoogle Scholar
  65. Zingsheim HP, Barrantes FJ, Frank J et al (1982) Direct structural localization of two toxin-recognition sites on an ACh receptor protein. Nature 299:81–84CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Laboratory of Protein Synthesis and Expression, Institute for Protein ResearchOsaka UniversitySuitaJapan

Personalised recommendations