Identifying innovative binders for the purification of secretory immunoglobulin A from complex matrices

Background.

Secretory immunoglobulin A (sIgA) are molecules of the adaptive immune system naturally occurring on mucosal surfaces. The dimeric structure sIgA makes them substantially more potent in neutralizing invading pathogens than other antibody classes such as monomeric IgG (Sun, 2021). However, whereas the purification of IgGs is straightforward, the isolation of sIgA is extremely challenging because no well-established affinity ligands are available. For example, typical resin capacities are <10 g L-1 at costs of >50,000 € L-1 in case of a protein M ligand and other resins exhibiting similar performance. Moreover, typical molecules binding sIgA are not selective for fully assembled sIgA that consist of 10 individual polypeptides of four different types. Accordingly, there is a substantial need for innovative ligands that facilitate selective binding to different domains of sIgA to ensure purification of fully-assembled molecules.

Hyptheses.

Binding of sIgA to peptides can be used to create innovative affinity ligands.

Methods.

Protein sequences known to interact with the different domains and polypeptides of sIgA will be identified based on literature search. The sequences will be diversified using methods such as error-prone PCR (Laurent, 2021) and will subsequently be expressed on the surface of yeast, resulting in highly diverse (~109) yeast display libraries (Zajc, 2020). Relevant sIgA will be taken from existing BOKU projects (Sun, 2021). Yeast displayed randomly mutated ligand candidates will be screened for optimal stability and affinity (Teufl, 2022). The resulting ligands will be synthesized in a suitable expression system like E. coli or plant cells and will be used to create novel affinity resins (Ruhl, 2018). These resins can either be built on a single ligand or a combination of ligands targeting different domains of sIgA. In the latter case, ligand immobilization will be tested for different stoichiometries as well as for different spatial patterns. The resins will then be used to capture sIgA from authentic, complex feedstocks such as cell culture supernatant or plant extract. Based on these results, the resin operation (e.g. necessary elution conditions) and performance (e.g. number of application cycles, capacity) will be assessed.

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