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Classical Complement Pathway Components C1r and C1s: Purification from Human Serum and in Recombinant Form and Functional Characterization

  • Véronique Rossi
  • Isabelle Bally
  • Monique Lacroix
  • Gérard J. Arlaud
  • Nicole M. Thielens
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1100)

Abstract

C1r and C1s are the proteases responsible for the activation and proteolytic activity of the C1 complex of the classical complement pathway, respectively. They are assembled into a Ca2+-dependent C1s–C1r–C1r–C1s tetramer which in turn associates with the recognition protein C1q. The C1 complex circulates in serum as a zymogen and is activated upon binding of C1q to appropriate targets, such as antigen–antibody complexes. This property is used for the purification of C1r and C1s from human serum after binding of C1 to insoluble immune complexes. Disruption of the bound C1 complex by EDTA releases C1r and C1s which are further separated by ion-exchange chromatography; both proteins can be reassembled in the presence of calcium ions and the reconstituted tetramer isolated by gel filtration. In this chapter, we describe the purification of the activated and proenzyme forms of C1r and C1s and of the proenzyme C1s–C1r–C1r–C1s tetramer as well as methods for their biochemical and functional characterization. The production of recombinant C1s and of the proenzyme tetramer in a baculovirus–insect cell system, and their purification by affinity chromatography is also presented.

Key words

Complement protease C1r C1s Purification Activation Enzymatic activity C1 reconstitution 

Notes

Acknowledgments

We thank Thomas Lunardi and Sarah Ancelet for help in preparing the figures. Part of the described work was supported by the Agence Nationale de la Recherche (grants ANR-05-MIIM-023-01 and ANR-09-PIRI-0021).

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Véronique Rossi
    • 1
  • Isabelle Bally
    • 1
  • Monique Lacroix
    • 1
  • Gérard J. Arlaud
    • 1
  • Nicole M. Thielens
    • 1
  1. 1.CEA, DSV, Institut de Biologie Structurale (IBS), CNRS, UMR 5075Université Joseph FourierGrenobleFrance

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