The rational design of affinity-attenuated OmCI for the purification of complement C5

Abstract

Complement component C5 is the target of the mAb eculizumab and is 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 with existing methods. However, breaking the high-affinity interaction requires conditions that 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 characterized mutants using a range of biophysical techniques, as well as functional assays. Our biophysical analyses suggest that 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 nondenaturing or activating to C5. We present the rational design, biophysical characterization, and experimental validation of affinity-reduced forms of OmCI as tool reagents to enable the affinity purification of C5.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies

“…Ninety-six-well ELISA plates (Nunc Maxisorp) were coated with a 2 μg/mL solution of C5, purified from serum as previously described [ 47 ], in carbonate-bicarbonate buffer (Sigma). All washing steps comprised 4 wash cycles with PBS, 0.05% Tween 20.…”

“…Using a Biacore 8K (GE Healthcare), C5 purified from serum [ 47 ] was immobilized on a CM5 chip by amine coupling. For PGT121 fusion proteins, flow cells were activated using a minimal immobilisation protocol: EDC/NHS was mixed at 1:2 ratio (flow rate, 10 μL/min; contact time, 30 s).…”

“…C5 purified from serum [ 47 ] was labelled with an amine reactive terbium chelate (molecular probes, Life Technologies), as per the manufacturer’s instructions. Briefly, C5 at 1.15 mg/mL was buffer exchanged into a 50 mM Bicine, 100 mM NaCl (pH 8.2) buffer using a Zeba column (Thermo Scientific).…”

The allosteric modulation of Complement C5 by knob domain peptides

“…Complement proteins. Human C5 was affinity purified using an E141A, H164A OmCI column (Macpherson et al, 2018). Briefly, human serum (TCS biosciences) was diluted 1:1 (v/v) with PBS, 20 mM EDTA and applied to a 5 mL Hi-Trap NHS column (GE Healthcare), which contained 20 mg of E141A H164A OmCI protein, at a rate of 1 mL/minute.…”

Chemical Equilibrium at the Tick–Host Feeding Interface:A Critical Examination of Biological Relevance in Hematophagous Behavior

“…These inhibitors have K D values in the low nM range (Table 1). A recent study that used site-specific immobilization of OMCI and multicycle kinetics indicated that the K D may even be in the low pM ranges indicating that assay design can affect estimations of affinity (Macpherson et al, 2018). These inhibitors also show quite high expected concentrations at the feeding site, which is mostly due to their functioning as kratagonists (section below).…”