The rational design of affinity-attenuated OmCI for the purification of complement C5
| Autor: | Jean M. H. van den Elsen, Bruce Carrington, Neesha Dedi, Xiaofeng Liu, Alastair D. G. Lawson, Sam Heywood, Jeffery Kennedy, Alex Macpherson, Oliver Durrant |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
medicine.drug_class Mutagenesis (molecular biology technique) Monoclonal antibody Biochemistry 03 medical and health sciences complement component C5 Affinity chromatography biophysics protein purification Drug Discovery Protein purification medicine Animals Humans complement OmCI Molecular Biology complement system Immune Evasion Complement component 5 Binding Sites Tandem Affinity Purification Chemistry Drug discovery Methods and Resources Rational design Complement C5 Cell Biology Complement system 030104 developmental biology Drug Design Mutagenesis Site-Directed mutagenesis Protein Binding |
| Zdroj: | The Journal of Biological Chemistry Macpherson, A, Liu, X, Dedi, N, Kennedy, J, Carrington, B, Durrant, O, Heywood, S, van den Elsen, J & Lawson, A D G 2018, ' The rational design of affinity attenuated OmCI for the purification of Complement C5 ', The Journal of biological chemistry, vol. 293, no. 36, pp. 14112-14121 . https://doi.org/10.1074/jbc.RA118.004043 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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