Identification and Characterization of Borrelia burgdorferi Complement-Binding Proteins

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1690)

Abstract

Acquisition of host-derived proteins possessing key regulatory function is a hallmark of Borrelia burgdorferi, and an important step to successfully infect the human host, inhibiting activation of complement as innate immunity’s first line of defense. Hence, the identification and characterization of interacting ligands is a prerequisite to gain deeper insights into the molecular principles of how spirochetes overcome the detrimental effects of complement. Far western blotting enables the detection of protein-protein interactions in vitro using cell lysates containing the prey proteins and purified complement proteins or human serum as a source for soluble complement proteins as bait proteins. Here, the methodology for the detection and characterization of Borrelia-derived proteins interacting with complement regulator Factor H is described, including the preparation of whole cell lysates, the separation of proteins by Tris-Tricine SDS-PAGE, the transfer of the proteins to nitrocellulose membranes, and the detection of Factor H-interacting proteins by Far western.

Key words

Borrelia Spirochete Lyme disease Complement Factor H Far western Tricine SDS-PAGE 

References

  1. 1.
    Edmondson DG, Dent SY (2001) Identification of protein interactions by far western analysis. Curr Protoc Protein Sci Chapter 19:Unit 19 17Google Scholar
  2. 2.
    Wu Y, Li Q, Chen XZ (2007) Detecting protein-protein interactions by far western blotting. Nat Protoc 2:3278–3284CrossRefPubMedGoogle Scholar
  3. 3.
    Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22CrossRefPubMedGoogle Scholar
  4. 4.
    Schagger H, Von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  5. 5.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  6. 6.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kraiczy P, Skerka C, Brade V et al (2001) Further characterization of complement regulator-acquiring surface proteins of Borrelia burgdorferi. Infect Immun 69:7800–7809CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kraiczy P, Skerka C, Kirschfink M et al (2001) Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and factor H. Eur J Immunol 31:1674–1684CrossRefPubMedGoogle Scholar
  9. 9.
    Alitalo A, Meri T, Comstedt P et al (2005) Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis. Eur J Immunol 35:3043–3053CrossRefPubMedGoogle Scholar
  10. 10.
    Bhide MR, Escudero R, Camafeita E et al (2009) Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human. BMC Res Notes 2:134Google Scholar
  11. 11.
    Prodinger WM, Hellwage J, Spruth M et al (1998) The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins. Biochem J 331:41–47CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Institute of Medical Microbiology and Infection ControlUniversity Hospital of FrankfurtFrankfurtGermany

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