Biaffin GmbH & Co. KG

 

Biaffin社は、Biacore™ SPR (表面プラズモン共鳴)技術を利用したアッセイで長年の経験と実績を持ち、様々な分子間の相互作用を、Label Freeかつリアルタイムで解析するサービスを提供しています。

Biacore™ SPR 技術を用いて、下記分子間等での相互作用解析が可能です。
  • 抗体-抗原
  • タンパク質-タンパク質
  • タンパク質-ペプチド
  • 低分子化合物-ターゲット (キナーゼ)

    Biaffin社では、タンパク質、ペプチド、DNA/RNA、糖タンパク質、脂質、低分子化合物等を、対象とする分子に適した方法でセンサーチップへ固定化し、温度、濃度、基質特異性等の反応条件等の様々な目的に応じて、下記各種Biacore™装置を用いたアッセイを行います。
  • Biacore™ 2000
  • Biacore™ S51
  • Biacore™ T100 / T200
  • Biacore™ X
    		•
    Sensorgram of an biomolecular interaction analysis
    The figure shows the interaction of an analyte in solution with its respective ligand immobilised on the sensor surface. Dissociation was initiated by switching to running buffer. Since the dissociation rate is slow the surface has to be regenerated with an appropriate elution buffer disrupting the complex. In this case the regeneration is complete and does not affect the binding activity of the immobilised ligand.

    《サービス一覧》
     
    《サービス概要》
    ◆ Antibody Characterisation
     
  • 抗原とのアフィニティ評価やカイネティクス解析を行います。
  • 反応条件や温度の違いによる抗原-抗体の相互作用への影響の解析が可能です。
  • 薬剤処理の有無による血清中の抗体の評価も可能です。
  • リコンビナントの抗原のQCにも有用です。
     
    Qualitative antibody characterisation: different binding characteristics of one antigen binding to a monoclonal or polyclonal antibody    		  
    Quantitative analysis of S. aureus bacterial antigen,
    KD= 4.8 nM
    [Oesterreich et al. Hum Vaccin Immunother. 2014]

    Pairwise Epitope Mapping / Identification of antibody isotypes     		   
    Immunoreactivity
    Identification of higher antibody concentrations in complex serum samples
     
    • ◆ Protein interaction
       タンパク質-タンパク質、タンパク質-リガンド等の相互作用を、定性的あるいは定量的に解析します。

    Isoform-specific binding of PKA regulatory subunits to the catalytic subunit, different association/dissociation kinetics (alpha vs. beta), influence of metal ions and nucleotides on binding kinetics, cooperative effects due to dimerisation (monomer vs. dimer)    		  
    Kinetic analysis of peptide inhibitor HT31 binding to PKA RII alpha
     
    Inhibitor PKI interaction with the closed (left, in the presence of ATP/Mg) or open (right, with ETDA) form of PKA
    Interaction analysis of the heatstable protein kinase inhibitor PKI immobilised on a Biacore sensor surface with the catalytic subunit of cAMP-dependent protein kinase PKA-C(alpha) as analyte in solution. Assuming a 1:1 binding model the non linear data analysis after subtraction of the reference signal yielded the following rate constants [Zimmermann et al., J. Biol. Chem. (1999), 274, 5370-5378]:
    kass = 1.5 x106 M-1 s-1; kdiss = 7.6 x 10-4 s-1; KD = 0.5 nM In the absence of ATP/Mg in a running buffer containing ETDA, PKA changes its conformation from a closed to an open state, resulting in a completely different binding kinetics for PKI. Here the binding affinity is lowered by a factor of 720 to KD = 360 nM.

    Different concentrations of protease cathepsin D binding to a coupled inhibitor    		  
    The solution competition assay of cathepsin D with the protease inhibitor reveals an EC50 = 0.1 nM.
    ◆ Kinascreen™ SPR services
      低分子化合物のキナーゼに対する結合速度・解離速度・解離定数等、カイネティクス解析が可能です。
    現在、下記キナーゼについてSPRアッセイが利用可能です。その他のターゲットについては、お問い合わせください。
  • EGF-R
  • ERBB4
  • ERK1 (MAPK3)
  • FGF-R1
  • IGF1-R
  • JNK2 (MAPK9)
  • LYN
  • c-MET
  • p38alpha (MAPK14)
  • p38beta (MAPK11)
  • p38gamma (MAPK12)
  • p38delta (MAPK13)
  • PKA (catalytic and regulatory subunits)
  • PKB/AKT
  • SRC
  • YES

    • Real-time kinetic analysis of general kinase inhibitor staurosporine binding to PKA Cα using surface plasmon resonance.    		  
    Real-time kinetic analysis of kinase inhibitor Dasatinib binding to LYN using surface plasmon resonance.

    Real-time kinetic analysis of kinase inhibitor SB203580 binding to p38α (left) and p38β (right) using surface plasmon resonance.
    ◆ Further Applications
     
    • Drug Development
      • ADME (interaction with the most abundant plasma proteins)
      • Hit validation and lead optimisation
      • Identification of unknown binding partners
      • Target validation
    • Thermodynamic Interaction analysis
      • Interaction analysis over a range of temperatures
      • Kinetic data expressed in thermodynamic terms
      • Evaluation of transistion state thermodynamic parameters
    • Concentration analysis
      • Calibration curve based assays (Direct binding assay / Indirect or competition assay)
      • Calibration Free Concentration Analysis (CFCA)
    • Nucleic Acid Interactions
    • DNA-DNA Interactions (hibridisation temp., length of nucleic acid, base pair substitutions)
    上記アッセイ系の他、各種カスタムアッセイについては、お問い合わせください。
    《参考文献》
     
    1. Oesterreich B, Lorenz B, Schmitter T, Kontermann R, Zenn M, Zimmermann B, Haake M, Lorenz U, Ohlsen K. "Characterization of the biological anti-staphylococcal functionality of hUK-66 IgG1, a humanized monoclonal antibody as substantial component for an immunotherapeutic approach." Hum Vaccin Immunother. 2014 Feb 4;10(4)
    2. Chaikuad A, Keates T, Vincke C, Kaufholz M, Zenn M, Zimmermann B, Gutiérrez C, Zhang RG, Hatzos-Skintges C, Joachimiak A, Muyldermans S, Herberg FW, Knapp S, Müller S. "Structure of Cyclin G associated kinase (GAK) Trapped in different Conformations using Nanobodies." Biochem J. 2014 Jan 17.
    3. Kummer L, Hsu CW, Dagliyan O, Macnevin C, Kaufholz M, Zimmermann B, Dokholyan NV, Hahn KM, Plückthun A. "Knowledge-Based Design of a Biosensor to Quantify Localized ERK Activation in Living Cells." Chem Biol. 2013 Jun 20;20(6):847-56
    4. Brown KM, Day JP, Huston E, Zimmermann B, Hampel K, Christian F, Romano D, Terhzaz S, Lee LC, Willis MJ, Morton DB, Beavo JA, Shimizu-Albergine M, Davies SA, Kolch W, Houslay MD, Baillie GS (2013) "Phosphodiesterase-8A binds to and regulates Raf-1 kinase." Proc Natl Acad Sci U S A. 2013 Mar 18
    5. Kummer L, Parizek P, Rube P, Millgramm B, Prinz A, Mittl PR, Kaufholz M, Zimmermann B, Herberg FW, Plückthun A. (2012) "Structural and functional analysis of phosphorylation-specific binders of the kinase ERK" Proc Natl Acad Sci USA 2012 Jul 27
    6. Munari F, Soeroes S, Zenn HM, Schomburg A, Kost N, Schroeder S, Klingberg R, Rezaei-Ghaleh N, Stuetzer A, Gelato KA, Walla PJ, Becker S, Schwarzer D, Zimmermann B, Fischle W, Zweckstetter M. "Methylation of K9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1beta with the nucleosome" (2012) J Biol Chem. 2012 Jul 19
    7. Grancha S, Ortiz AM, Maranon C, Hampel K, Moret A, Zimmermann B, Jorquera JI, Aznar JA (2012) "Kinetics of the interaction between anti-FVIII antibodies and FVIII from therapeutic concentrates, with and without von Willebrand factor, assessed by surface plasmon resonance" Haemophilia, May 30, 2012
    8. Düvel J, Bertinetti D, Möller S, Schwede F, Morr M, Wissing J, Radamm L, Zimmermann B, Genieser HG, Jänsch L, Herberg FW, Häussler S (2011) "A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa." J Microbiol Methods
    9. Schmidt J, Müsken M, Becker T, Magnowska Z, Bertinetti D, Möller S, Zimmermann B, Herberg FW, Jänsch L, Häussler S (2011) "The Pseudomonas aeruginosa Chemotaxis Methyltransferase CheR1 Impacts on Bacterial Surface Sampling" PLoS One 6, 3
    10. Christian F, Szaszak M, Drewianka S, Hampel K, Herberg FW, Zimmermann B, Rosenthal W, Klussmann E (2011) "Small molecule AKAP/PKA interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes" J Biol Chem. 2011 Jan 5.
    11. Fender A, Elf J, Hampel K, Zimmermann B, Wagner EG (2010) "RNAs actively cycle on the Sm-like protein Hfq" Genes Dev. 24(23):2621-6
    12. Lorenz U, Lorenz B, Schmitter T, Streker K, Erck C, Wehland J, Nickel J, Zimmermann B, Ohlsen K. (2010) "Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy" Antimicrob Agents Chemother. 2010 Oct 18
    13. Diskar M, Zenn HM, Kaupisch A, Kaufholz M, Brockmeyer S, Sohmen D, Berrera M, Zaccolo M, Boshart M, Herberg FW , Prinz A. (2010) Regulation of cAMP-dependent protein kinases: the human Protein Kinase X (PrKX) reveals the role of the catalytic subunit alpha H alpha I loop." J Biol Chem. 2010 Sept 6
    14. Hundsrucker C, Skroblin P, Christian F, Zenn HM, Popara V, Joshi M, Eichhorst J, Wiesner B, Herberg FW , Reif B, Rosenthal W, Klussmann E. (2010) "Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein." J Biol Chem. 285(8):5507-21
    15. Zenn HM, Hutschenreiter S and Herberg FW (2010) "Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis" Antibody Engineering, Part V: 667-681
    16. Bertinetti D, Schweinsberg S, Hanke SE, Schwede F, Bertinetti O, Drewianka S, Genieser HG, Herberg FW (2009) "Chemical tools selectively target components of the PKA system." BMC Chem Biol. 9:3
    17. Zimmermann B, Schweinsberg S, Drewianka S, Herberg FW (2008) "Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA." Biochem J. 413(1):93-101
    18. Schweinsberg S, Moll D, Burghardt NC, Hahnefeld C, Schwede F, Zimmermann B, Drewianka S, Werner L, Kleinjung F, Genieser HG, Schuchhardt J, Herberg FW (2008) "Systematic interpretation of cyclic nucleotide binding studies using KinetXBase" Proteomics 8(6):1212-20
    19. Mirow N, Zimmermann B, Maleszka A, Knobl H, Tenderich G, Koerfer R, Herberg FW (2007) "Plasma Protein Binding Properties to Immobilized Heparin and Heparin-Albumin Conjugate" Artif Organs 31(6):466-71
    20. Seitz H, Hutschenreiter S, Hultschig C, Zeilinger C, Zimmermann B, Kleinjung F, Schuchhardt J, Eickhoff H, Herberg FW (2006) "Differential binding studies applying functional protein microarrays and surface plasmon resonance" Proteomics 6(19):5132-9
    21. Gesellchen, F., Prinz, A., Zimmermann B., Herberg F.W. (2006). "Quantification of cAMP antagonist action in vitro and in living cells." Eur J Cell Biol. 85(7):663-72
    22. Hundsrucker C, Krause G, Zimmermann B, Diekmann O, Herberg FW, Scott JD, Rosenthal W, Klussmann E et al. (2006) "High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides" Biochem J. 396(2):297-306
    23. Moll D., Zimmermann B., Gesellchen F., Herberg F.W. (2006) “Current developments for the in vitro characterization of protein interactions”. Proteomics in Drug Research. Edit. by K. Marcus, K. Stühler, Warscheidet et al. Weinheim, Wiley-VCH
    24. Moll D., Prinz A., Gesellchen F., Drewianka S., Zimmermann B. and Herberg F.W. (2006) "Biomolecular interaction analysis in functional proteomics" J. Neural Transmission
    25. Gesellchen F., Zimmermann B., and Herberg F.W. (2005) "Direct Optical Detection of Protein–Ligand Interactions" Humana Press, Methods in Molecular Biology, Protein–Ligand Interactions: Methods and Applications, Vol. 305, chapter 2, 17-46
    26. Ramanavicius A., Herberg F.W. et al. (2005). "Biomedical Application of Surface Plasmon Resonance Biosensors" Acta Medica Lithuania 12 (3): 1–9
    27. Thierse H.J., Moulon C., Allespach Y., Zimmermann B., Doetze A., Kuppig S., Wild D., Herberg F., Weltzien H.U. (2004) “Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation” J Immunol. 172(3):1926-34
    28. Hahnefeld C., Drewianka S., Herberg F.W. (2004) “Determination of kinetic data using surface plasmon resonance biosensors” Methods Mol Med. 94:299-320
    29. Yaqub S., Abrahamsen H., Zimmermann B., Kholod N., Torgersen K.M., Mustelin T., Herberg F.W., Tasken K., Vang T. (2003) “Activation of c-terminal Src kinase (Csk) by phosphorylation at Ser-364 depends on the Csk SH3 domain”, Biochem. J. 372, 271-278
    30. Zimmermann B., Hahnefeld C., Herberg F.W. (2002) “Applications of biomolecular interaction analysis in drug development”, Targets 2 (1) 66-73
    31. Wang X., Hu B., Zimmermann B., Kilimann M.W. (2001) “Rim1 and Rabphilin-3 bind Rab3-GTP by composite determinants partially related through N-terminal alpha-helix motifs”, J. Biol. Chem. 276 (35), 32480-8
    32. Herberg F.W. & Zimmermann B. (1999) “Analysis of protein kinase interactions using biomolecular interaction analysis”, Protein Phosphorylation, A Practical Approach, edited by D.G. Hardie, Oxford University Press
    33. Zimmermann B., Chiorini J.A., Kotin R.M., Herberg F.W. (1999) “PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I”, J. Biol.Chem. 274, 9, 5370-5378
    34. Herberg F.W., Doyle M.L., Cox S., Taylor S.S. (1999), “Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase”, Biochemistry 38(19):6352-60
    35. Gehrmann T., Gulkan H., Suer S., Herberg F.W., Balla A., Vereb G., Mayr G.W., Heilmeyer L.M. Jr (1999), “Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230”, Biochim. Biophys. Acta 1437(3):341-56
    36. Schliebs W., Saidowsky J., Agianian B., Dodt G., Herberg F.W., Kunau W.H. (1999), “Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14”, J. Biol. Chem. 274(9):5666-73
    37. Zimmermann B. & Herberg F.W. (1998) “Interactions of 6xHis-tagged protein kinase A catalytic subunit examined using Ni-NTA Magnetic agarose beads”, QIAGEN News 4/98, 9-10
    38. Chiorini J.A., Zimmermann B., Yang L., Smith R.H., Ahearn A., Herberg F.W., Kotin R.M. (1998) “Inhibition of PrKX, a novel protein kinase, and the cAMP-dependent protein kinase PKA, by the regulatory proteins of Adeno-associated virus type 2”, Mol. Cell. Biol. 18: 5921-5929
    39. Reiffert S.U., Jaquet K., Heilmeyer L.M.G. jr, Herberg F.W. (1998), “Stepwise Subunit Interaction Changes by Mono- and Bisphosphorylation of Cardiac Troponin I”, Biochemistry 37 (39), 13516-13525 (1998)
    40. Jaeger W., Haase B., Lutz V., Herberg F.W. and Zimmermann B. (1997) “Biomolekulare Interaktionsanalyse (BIA) mit SPR-Biosensortechnologie”, Biospektrum 3(4), 8-87
    41. Herberg F.W., Zimmermann B., McGlone M., Taylor S.S. (1997) “Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface”, Protein Science 6, 569-79
    42. Herberg F.W., Taylor S.S., Dostmann W.R. (1996), “Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase”, Biochemistry 35(9), 2934-2942
    43. Wild N., Herberg F.W., Hofmann F., Dostmann W.R. (1995), “Expression of a chimeric, cGMP-sensitive regulatory subunit of the cAMP-dependent protein kinase type I alpha”, FEBS Lett. 374(3), 356-362
    44. Su Y., Dostmann W.R., Herberg F.W., Durick K., Xuong N.H., Ten Eyck L., Taylor S.S., Varughese K.I. (1995) “Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains”, Science 269(5225), 807-813
    45. Herberg F.W., Dostmann W.R., Zorn M., Davis S.J., Taylor S.S. (1994) “Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation”, Biochemistry 33(23), 7485-7494
    46. Herberg F.W., Taylor S.S. (1993), “Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein-protein interactions”, Biochemistry 32(50), 14015-14022 (1993)
    47. Herberg F.W., Bell S.M., Taylor S.S. (1993) “Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states”, Protein Eng. 6(7), 771-777
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