Advertisement

Site-Specific Protein Labeling in the Pharmaceutical Industry: Experiences from Novartis Drug Discovery

  • Lukas LederEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)

Abstract

Chemically modified proteins play an important role in several fields of pharmaceutical R&D, starting from various activities in drug discovery all the way down to biopharmaceuticals with improved properties such as antibody–drug conjugates. In the first part of the present chapter the significance and use of labeled proteins in biophysical methods, biochemical and cellular assays, in vivo imaging, and biopharmaceuticals is reviewed in general. In this context, the most relevant methods for site-specific modification of proteins and their application are also described. In the second part of the chapter, in-house (Novartis) results and experience with different techniques for selective protein labeling are discussed, with a focus on chemical or enzymatic (Avi-tag) biotinylation of proteins and their application in biophysical and biochemical assays. It can be concluded that while modern methods of site-specific protein labeling offer new possibilities for pharmaceutical R&D, classical methods are still the mainstay mainly due to being well established. However, site-specific protein labeling is expected to increase in importance, in particular for antibody–drug conjugates and other chemically modified biopharmaceuticals.

Key words

Biophysical methods Biochemical assay Cellular assays In vivo imaging Biopharmaceutical Antibody–drug conjugates Biotin ligase Avi-tag SNAP-tag Transglutaminase Lipoic acid ligase Click chemistry Sortase Phosphopantetheinyl transferase 

Notes

Acknowledgements

I would like to thank the following colleagues: Jutta Blank, Marjo, Goette, Christian Bergsdorf, and Rainer Kneuer who gave valuable input and discussion in terms of performed experiments and general approaches in their respective field of expertise.

References

  1. 1.
    Hinner MJ, Johnsson K (2010) How to obtain labeled proteins and what to do with them. Curr Opin Biotechnol 21(6):766–776PubMedCrossRefGoogle Scholar
  2. 2.
    Foley TL, Burkart MD (2007) Site-specific protein modification: advances and applications. Curr Opin Chem Biol 11(1):12–19PubMedCrossRefGoogle Scholar
  3. 3.
    Rashdian M, Dozier JK, Distefano MK (2013) Enzymatic labeling of proteins: techniques and approaches. Bioconjug Chem 24(8):1277–1294CrossRefGoogle Scholar
  4. 4.
    Tirat A, Freuler F, Stettler T, Mayr LM, Leder L (2006) Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins. Int J Biol Macromol 39(1–3):66–76PubMedCrossRefGoogle Scholar
  5. 5.
    Fernandez-Suarez M, Baruah H, Martinez-Hernandez L, Xie KT, Baskin JM, Bertozzi CR, Ting AY (2007) Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol 25(12):1483–1487PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Puthenveetil S, Liu DS, White KA, Thompson S, Ting AY (2009) Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J Am Chem Soc 131(45):16430–16438PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Yin J, Straight PD, McLoughlin SM, Zhou Z, Lin AJ, Golan DE, Kelleher NL, Kolter R, Walsh CT (2005) Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc Natl Acad Sci U S A 102(44):15815–15820PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Zhou Z, Cironi P, Lin AJ, Xu Y, Hrvatin S, Golan DE, Silver PA, Walsh CT, Yin J (2007) Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem Biol 2(5):337–346PubMedCrossRefGoogle Scholar
  9. 9.
    Lin CW, Ting AY (2006) Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J Am Chem Soc 128(14):4542–4543PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Jager M, Nir E, Weiss S (2006) Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification. Protein Sci 15(3):640–646PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(11):707–708PubMedCrossRefGoogle Scholar
  12. 12.
    Antos JM, Chew GL, Guimaraes CP, Yoder NC, Grotenbreg GM, Popp MW, Ploegh HL (2009) Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J Am Chem Soc 131:10800–10801PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Young TS, Schultz PG (2010) Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem 285(15):11039–11044PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Ou WJ, Uno T, Chiu HP, Grunewald J, Cellitti SE, Crossgrove T, Hao XS, Fan Q, Quinn LL, Patterson P et al (2011) Site-specific protein modifications through pyrroline-carboxy-lysine residues. Proc Natl Acad Sci U S A 108(26):10437–10442PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Danielson UH (2009) Integrating surface plasmon resonance biosensor-based interaction kinetic analyses into the lead discovery and optimization process. Future Med Chem 1(8):1399–1414PubMedCrossRefGoogle Scholar
  16. 16.
    Huber W (2011) SPR-based direct binding assays in drug discovery. In: Cooper M, Mayr LM (eds) Label-free technologies for drug discovery. John Wiley & Sons, Ltd., ChichesterGoogle Scholar
  17. 17.
    Syguda A, Kerstan A, Ladnorg T, Stuben F, Woll C, Herrmann C (2012) Immobilization of biotinylated hGBP1 in a defined orientation on surfaces is crucial for uniform interaction with analyte proteins and catalytic activity. Langmuir 28(15):6411–6418PubMedCrossRefGoogle Scholar
  18. 18.
    Hutsell SQ, Kimple RJ, Siderovski DP, Willard FS, Kimple AJ (2010) High-affinity immobilization of proteins using biotin- and GST-based coupling strategies. Methods Mol Biol 627:75–90PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Huang ZH, Hwang P, Watson DS, Cao LM, Szoka FC (2009) Tris-nitrilotriacetic acids of subnanomolar affinity toward hexahistidine tagged molecules. Bioconjug Chem 20(8):1667–1672PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Chen YX, Triola G, Waldmann H (2011) Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins. Acc Chem Res 44(9):762–773PubMedCrossRefGoogle Scholar
  21. 21.
    Wong LS, Khan F, Micklefield J (2009) Selective covalent protein immobilization: strategies and applications. Chem Rev 109(9):4025–4053PubMedCrossRefGoogle Scholar
  22. 22.
    Wammes AE, Fischer MJ, de Mol NJ, van Eldijk MB, Rutjes FP, van Hest JC, van Delft FL (2013) Site-specific peptide and protein immobilization on surface plasmon resonance chips via strain-promoted cycloaddition. Lab Chip 13(10):1863–1867PubMedCrossRefGoogle Scholar
  23. 23.
    Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51–76PubMedCrossRefGoogle Scholar
  24. 24.
    Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B (2009) HTRF: a technology tailored for drug discovery—a review of theoretical aspects and recent applications. Curr Chem Genomics 3:22–32PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Gotoh Y, Nagata H, Kase H, Shimonishi M, Ido M (2010) A homogeneous time-resolved fluorescence-based high-throughput screening system for discovery of inhibitors of IKKbeta-NEMO interaction. Anal Biochem 405(1):19–27PubMedCrossRefGoogle Scholar
  26. 26.
    Nakamura K, Zawistowski JS, Hughes MA, Sexton JZ, Yeh LA, Johnson GL, Scott JE (2008) Homogeneous time-resolved fluorescence resonance energy transfer assay for measurement of Phox/Bem1p (PB1) domain heterodimerization. J Biomol Screen 13(5):396–405PubMedCrossRefGoogle Scholar
  27. 27.
    Kim B, Tarchevskaya SS, Eggel A, Vogel M, Jardetzky TS (2012) A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions. Anal Biochem 431(2):84–89PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Mollwitz B, Brunk E, Schmitt S, Pojer F, Bannwarth M, Schiltz M, Rothlisberger U, Johnsson K (2012) Directed evolution of the suicide protein O6-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability. Biochemistry 51(5):986–994PubMedCrossRefGoogle Scholar
  29. 29.
    Gautier A, Juillerat A, Heinis C, Correa IR, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cell. Chem Biol 15(2):128–136PubMedCrossRefGoogle Scholar
  30. 30.
    Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382PubMedCrossRefGoogle Scholar
  31. 31.
    Shi X, Jung Y, Lin LJ, Liu C, Wu C, Cann IK, Ha T (2012) Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging. Nat Methods 9(5):499–503PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Lemke EA (2013) Site-specific labeling of proteins for single-molecule FRET measurements using genetically encoded ketone functionalities. In: Mark SS (ed) Bioconjugation protocols, vol 751, Methods in molecular biology. Humana Press, New York, NY, pp 3–15CrossRefGoogle Scholar
  33. 33.
    Kim J, Seo MH, Lee S, Cho K, Yang A, Woo K, Kim HS, Park HS (2013) Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis. Anal Chem 85(3):1468–1474PubMedCrossRefGoogle Scholar
  34. 34.
    Lian LY, Middleton DA (2001) Labelling approaches for protein structural studies by solution-state and solid-state NMR. Prog Nucl Mag Res Sp 39(3):171–190CrossRefGoogle Scholar
  35. 35.
    Hoogstraten CG, Johnson JE (2008) Metabolic labeling: taking advantage of bacterial pathways to prepare spectroscopically useful isotope patterns in proteins and nucleic acids. Concept Magn Reson 32A(1):34–55CrossRefGoogle Scholar
  36. 36.
    Weigelt J, Wikstrom M, Schultz J, van Dongen MJ (2002) Site-selective labeling strategies for screening by NMR. Comb Chem High Throughput Screen 5(8):623–630PubMedCrossRefGoogle Scholar
  37. 37.
    Jahnke W, Rudisser S, Zurini M (2001) Spin label enhanced NMR screening. J Am Chem Soc 123(13):3149–3150PubMedCrossRefGoogle Scholar
  38. 38.
    Jones DH, Cellitti SE, Hao X, Zhang Q, Jahnz M, Summerer D, Schultz PG, Uno T, Geierstanger BH (2010) Site-specific labeling of proteins with NMR-active unnatural amino acids. J Biomol NMR 46(1):89–100PubMedCrossRefGoogle Scholar
  39. 39.
    Yang Y, Li QF, Cao C, Huang F, Su XC (2013) Site-specific labeling of proteins with a chemically stable, high-affinity tag for protein study. Chemistry 19(3):1097–1103PubMedCrossRefGoogle Scholar
  40. 40.
    Shimba N, Yamada N, Yokoyama K, Suzuki E (2002) Enzymatic labeling of arbitrary proteins. Anal Biochem 301(1):123–127PubMedCrossRefGoogle Scholar
  41. 41.
    Crivat G, Taraska JW (2012) Imaging proteins inside cells with fluorescent tags. Trends Biotechnol 30(1):8–16PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90(3):1103–1163PubMedCrossRefGoogle Scholar
  43. 43.
    Jing C, Cornish VW (2011) Chemical tags for labeling proteins inside living cells. Acc Chem Res 44(9):784–792PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Uttamapinant C, White KA, Baruah H, Thompson S, Fernandez-Suarez M, Puthenveetil S, Ting AY (2010) A fluorophore ligase for site-specific protein labeling inside living cells. Proc Natl Acad Sci U S A 107(24):10914–10919PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Reymond L, Lukinavicius G, Umezawa K, Maurel D, Brun MA, Masharina A, Bojkowska K, Mollwitz B, Schena A, Griss R et al (2011) Visualizing biochemical activities in living cells through chemistry. Chimia 65(11):868–871PubMedCrossRefGoogle Scholar
  46. 46.
    Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A 104(43):16793–16797PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Lang K, Davis L, Torres-Kolbus J, Chou C, Deiters A, Chin JW (2012) Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 4(4):298–304PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Seitchik JL, Peeler JC, Taylor MT, Blackman ML, Rhoads TW, Cooley RB, Refakis C, Fox JM, Mehl RA (2012) Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctene. J Am Chem Soc 134:2898–2901PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Ripoll J, Ntziachristos V, Cannet C, Babin AL, Kneuer R, Gremlich HU, Beckmann N (2008) Investigating pharmacology in vivo using magnetic resonance and optical imaging. Drugs R&D 9(5):277–306CrossRefGoogle Scholar
  50. 50.
    Kneuer R, Gremlich HU, Beckmann N, Jetzfellner T, Ntziachristos V (2012) In vivo fluorescence optical and multi-modal imaging in pharmacological research: from chemistry to therapy monitoring, RSC Drug Discovery Series. RSC Publishing, Cambridge, pp 343–370Google Scholar
  51. 51.
    Wu AM, Olafsen T (2008) Antibodies for molecular imaging of cancer. Cancer J 14(3):191–197PubMedCrossRefGoogle Scholar
  52. 52.
    van Dongen GA, Visser GW, Lub-de Hooge MN, de Vries EG, Perk LR (2007) Immuno-PET: a navigator in monoclonal antibody development and applications. Oncologist 12(12):1379–1389PubMedCrossRefGoogle Scholar
  53. 53.
    Wang H, Chen X (2008) Site-specifically modified fusion proteins for molecular imaging. Front Biosci 13:1716–1732PubMedCrossRefGoogle Scholar
  54. 54.
    Blankenberg FG, Backer MV, Levashova Z, Patel V, Backer JM (2006) In vivo tumor angiogenesis imaging with site-specific labeled (99 m)Tc-HYNIC-VEGF. Eur J Nucl Med Mol Imaging 33(7):841–848PubMedCrossRefGoogle Scholar
  55. 55.
    Backer MV, Levashova Z, Levenson R, Blankenberg FG, Backer JM (2008) Cysteine-containing fusion tag for site-specific conjugation of therapeutic and imaging agents to targeting protein. Methods Mol Biol 494:275–294PubMedCrossRefGoogle Scholar
  56. 56.
    Wållberg H, Grafström J, Cheng Q, Lu L, Martinsson Ahlzén HS, Samén E, Thorell JO, Johansson K, Dunås F, Olofsson MH, Stone-Elander S et al (2012) HER2-positive tumors imaged within 1 hour using a site-specifically 11C-labeled Sel-tagged affibody molecule. J Nucl Med 53(9):1446–1453PubMedCrossRefGoogle Scholar
  57. 57.
    Pardo A, Stöcker M, Kampmeier F, Melmer G, Fischer R, Thepen T, Barth S (2012) In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model. Cancer Immunol Immunother 61(10):1617–1625PubMedCrossRefGoogle Scholar
  58. 58.
    Kosaka N, Ogawa M, Choyke PL, Karassina N, Corona C, McDougall M, Lynch DT, Hoyt CC, Levenson RM, Los GV et al (2009) In vivo stable tumor-specific painting in various colors using dehalogenase-based protein-tag fluorescent ligand. Bioconjug Chem 20(7):1367–1374PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Molineux G (2003) Pegylation: engineering improved biopharmaceuticals for oncology. Pharmacotherapy 23(8 Pt 2):3S–8SPubMedCrossRefGoogle Scholar
  60. 60.
    Veronese FM, Mero A (2008) The impact of PEGylation on biological therapies. BioDrugs 22(5):315–329PubMedCrossRefGoogle Scholar
  61. 61.
    Molineux G (2004) The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta). Curr Pharm Des 10(11):1235–1244PubMedCrossRefGoogle Scholar
  62. 62.
    Yamamoto Y, Tsutsumi Y, Yoshioka Y, Nishibata T, Kobayashi K, Okamoto T, Mukai Y, Shimizu T, Nakagawa S, Nagata S et al (2003) Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity. Nat Biotechnol 21(5):546–552PubMedCrossRefGoogle Scholar
  63. 63.
    Rosendahl MS, Doherty DH, Smith DJ, Carlson SJ, Chlipala EA, Cox GN (2005) A long-acting, highly potent interferon alpha-2 conjugate created using site-specific PEGylation. Bioconjug Chem 16(1):200–207PubMedCrossRefGoogle Scholar
  64. 64.
    Qiu H, Boudanova E, Park A, Bird JJ, Honey DM, Zarazinski C, Greene B, Kingsbury JS, Boucher S, Pollock J et al (2013) Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action. Bioconjug Chem 24(3):408–418PubMedCrossRefGoogle Scholar
  65. 65.
    Sato H, Hayashi E, Yamada N, Yatagai M, Takahara Y (2001) Further studies on the site-specific protein modification by microbial transglutaminase. Bioconjug Chem 12(5):701–710PubMedCrossRefGoogle Scholar
  66. 66.
    Popp MW, Dougan SK, Chuang TY, Spoonera E, Ploegh HL (2010) Sortase-catalyzed transformations that improve the properties of cytokines. Proc Natl Acad Sci U S A 108:369–374Google Scholar
  67. 67.
    Kochendoerfer GG (2005) Site-specific polymer modification of therapeutic proteins. Curr Opin Chem Biol 9(6):555–560PubMedCrossRefGoogle Scholar
  68. 68.
    Katz J, Janik JE, Younes A (2011) Brentuximab Vedotin (SGN-35). Clin Cancer Res 17(20):6428–6436PubMedCrossRefGoogle Scholar
  69. 69.
    Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RV, Lutz RJ et al (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68(22):9280–9290PubMedCrossRefGoogle Scholar
  70. 70.
    Adair JR, Howard PW, Hartley JA, Williams DG, Chester KA (2012) Antibody-drug conjugates - a perfect synergy. Expert Opin Biol Ther 12(9):1191–1206PubMedCrossRefGoogle Scholar
  71. 71.
    Zolot RS, Basu S, Million RP (2013) Antibody-drug conjugates. Nat Rev Drug Discov 12(4):259–260PubMedCrossRefGoogle Scholar
  72. 72.
    Flygare JA, Pillow TH, Aristoff P (2013) Antibody-drug conjugates for the treatment of cancer. Chem Biol Drug Des 81(1):113–121PubMedCrossRefGoogle Scholar
  73. 73.
    Ricart AD (2011) Antibody-drug conjugates of calicheamicin derivative: gemtuzumab ozogamicin and inotuzumab ozogamicin. Clin Cancer Res 17(20):6417–6427PubMedCrossRefGoogle Scholar
  74. 74.
    Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932PubMedCrossRefGoogle Scholar
  75. 75.
    Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth JS, Santidrian AF, Stafin K et al (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 109(40):16101–16106PubMedCentralPubMedCrossRefGoogle Scholar
  76. 76.
    Tian F, Lu Y, Manibusan A, Sellers A, Tran H, Sun Y, Phuong T, Barnett R, Hehli B, Song F et al (2014) A general approach to site-specific antibody drug conjugates. Proc Natl Acad Sci U S A 111(5):1766–1771PubMedCentralPubMedCrossRefGoogle Scholar
  77. 77.
    Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, Ho WH, Farias S, Casas MG, Abdiche Y et al (2013) Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol 20(2):161–167PubMedCrossRefGoogle Scholar
  78. 78.
    Jeger S, Zimmermann K, Blanc A, Grunberg J, Honer M, Hunziker P, Struthers H, Schibli R (2010) Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew Chem Int Ed Engl 49(51):9995–9997PubMedCrossRefGoogle Scholar
  79. 79.
    Matz J, Chames P (2012) Phage display and selections on purified antigens. Methods Mol Biol 907:213–224PubMedCrossRefGoogle Scholar
  80. 80.
    Kruszynski M, Tsui P, Stowell N, Luo J, Nemeth JF, Das AM, Sweet R, Heavner GA (2006) Synthetic, site-specific biotinylated analogs of human MCP-1. J Pept Sci 12(5):354–360PubMedCrossRefGoogle Scholar
  81. 81.
    Scholle MD, Kriplani U, Pabon A, Sishtla K, Glucksman MJ, Kay BK (2006) Mapping protease substrates by using a biotinylated phage substrate library. Chembiochem 7(5):834–838PubMedCrossRefGoogle Scholar
  82. 82.
    Tirat A, Schilb A, Riou V, Leder L, Gerhartz B, Zimmermann J, Worpenberg S, Eidhoff U, Freuler F, Stettler T et al (2005) Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2. Anal Biochem 343(2):244–255PubMedCrossRefGoogle Scholar
  83. 83.
    Geierstanger BH, Grunewald J, Bursulaya B (2013) Use of phosphopantetheinyl transferase substrate peptides for site-specific labeling of immunoglobulins. PCT Int Appl. PCT/US2013/043684Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Center for Proteomic ChemistryNovartis Institutes for Biomedical ResearchBaselSwitzerland

Personalised recommendations