(471b) Engineering of the RTX Domain Using Phage-Assisted Continuous Evolution (PACE) for Biomolecular Recognition for Application in Affinity Chromatography.

The ever-increasing number of non-antibody therapeutic proteins approved by the FDA and the increased use of proteins in various industries pose new challenges and call for the development of a universal and easy-to-deploy purification platform. The repeats-in-toxins (RTX) domain is an intrinsically disordered protein that folds into a beta-roll upon binding calcium ions with eight mutable solvent-exposed amino acids on each face. We exploit the nature of the RTX domain to develop a calcium-dependent protein purification platform by engineering the surface of the folded domain to recover target proteins from the cell lysate selectively. We coupled site-directed mutagenesis with phage-assisted continuous evolution (PACE) to engineer the surface to bind biomolecules of interest. We developed selective mutants in less than 100 hours of evolution, a process that could take weeks, if not months, using other directed evolution methods. As a proof of concept, we evolved the RTX domain to bind two distinctly different proteins based on their secondary structures. The engineered RTX domains were able to bind the ZR domain (alpha-helix) and the cyan fluorescent protein (beta-sheets and loops) with nanomolar affinity. The proteins of interest were recovered from cell lysate using immobilized mutants of the RTX domain and released using mild-elution conditions (pH 7.4) in a calcium-dependent manner.