Abstract
While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody–peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 μM), recovery and purity (64–71% and 86–91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.
Highlights
While still dominated by monoclonal antibodies, the landscape of therapeutic and diagnostic proteins recently witnessed the emergence of other species, in particular small-molecular-weight scaffolds [1,2], like adnectins [3], anticalins [4] DARPins [5], knottins [6], and affibodies [7]
A “one-bead one-peptide” (OBOP) library of linear peptides was initially built on hydroxymethylbenzoic acid (HMBA)-ChemMatrix resin following the “split-couple-and-recombine” (SCR) method described by Lam et al [45], and screened to discover affibody-binding peptide ligands by adapting selection methods developed by our group [37,38]
To impart a broad affibody-binding activity to the selected peptides, we adopted two model targets, namely, an anti-IgG [46,47] and an anti-human serum albumin (HSA) affibody [48]. These were each labeled with two fluorescent dyes, either the green AlexaFluor 488 (AF488) or the red AlexaFluor 594 (AF594), resulting in two orthogonal target pairs, namely, a green anti-IgG affibody and a red anti-HSA affibody, as well as a red anti-IgG affibody and a green anti-HSA affibody
Summary
While still dominated by monoclonal antibodies, the landscape of therapeutic and diagnostic proteins recently witnessed the emergence of other species, in particular small-molecular-weight scaffolds [1,2], like adnectins [3], anticalins [4] DARPins (designed ankyrin repeat proteins) [5], knottins [6], and affibodies [7]. The native Z domain was widely commercialized as an affinity ligand for the purification of antibodies by affinity chromatography [14], and it was extensively manipulated by combinatorial engineering and in vitro selection methods to obtain protein-binding affibodies [15]. These comprise 13 surface-displayed amino acids on helices 1 and 2 that are tailored to ensure selective binding of the target protein, while helix 3 and part of helix 1 are maintained constant (Table 1) [16]. The ability to display a functional amino acid (e.g., cysteine) on the C- or N-terminus of affibodies without affecting their structure and binding activity facilitates their conjugation to fluorescent probes or therapeutic payloads, or onto chromatographic materials or sensor hardware [13,19]
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