The rational design of affinity attenuated OmCI for the purification of Complement C5

Alex Macpherson, Xiaofeng Liu, Neesha Dedi, Jeffery Kennedy, Bruce Carrington, Oliver Durrant, Sam Heywood, Jean van den Elsen, Alastair D G Lawson

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Abstract

Complement component C5 is the target of the monoclonal antibody Eculizumab, and the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to, and potently inactivates, C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared to existing methods. However, breaking the high-affinity interaction requires conditions which risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site directed mutagenesis, and characterised mutants using a range of biophysical techniques as well as functional assays. Our biophysical analyses suggest the C5-OmCI interaction is complex with potential for multiple binding modes.We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease, or entirely abrogate, formation of the complex. The affinity attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions that are non-denaturing or activating to C5. We present the rational design, biophysical characterisation and experimental validation of affinity reduced forms of OmCI as tool reagents to enable the affinity purification of C5.

Original languageEnglish
Pages (from-to)14112-14121
Number of pages10
JournalThe Journal of biological chemistry
Volume293
Issue number36
Early online date20 Jul 2018
DOIs
Publication statusPublished - 7 Sep 2018

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Macpherson, A., Liu, X., Dedi, N., Kennedy, J., Carrington, B., Durrant, O., ... Lawson, A. D. G. (2018). The rational design of affinity attenuated OmCI for the purification of Complement C5. The Journal of biological chemistry, 293(36), 14112-14121. https://doi.org/10.1074/jbc.RA118.004043