The β-roll is a unique, allosterically–regulated peptide motif. This peptide is isolated from the repeats-in-toxin (RTX) domain and is intrinsically disordered in the absence of calcium. In calcium rich environments, the peptide binds Ca++ ions and folds into a β-roll secondary structure composed of two parallel β-sheet faces. We have extensively characterized this calcium responsive RTX domain and evaluated its potential as a new bioseparations platform in both non-chromatographic and affinity chromatography applications. We have developed a synthetic peptide, based on RTX domains, which undergoes calcium-responsive, reversible precipitation. This synthetic tag was appended to green fluorescent protein, β-lactamase and alcohol dehydrogenase. After protease cleavage downstream of the precipitating tag, pure and active target proteins were obtained by cycling precipitations before and after cleavage. This work demonstrates a new stimulus-responsive precipitating tag that can be used for efficient bioseparations using gentler conditions than existing alternatives, enabling purification of recombinant proteins from lysate in only a few minutes1. More recently, we have also shown that this β-roll peptide domain can serve as a new scaffold for engineering controllable biomolecular recognition. Mutant peptides were selected against lysozyme via ribosome display where we identified peptides with mid-nanomolar (63μM) dissociation constants. We showed that the mutant RTX peptide is capable of capturing its target in affinity chromatography experiments in the presence of calcium and captured target is easily eluted upon removal of the calcium ions, in a Catch and Release mechanism2. Currently we are constructing a new platform based on different RTX mutants where the precipitating RTX peptide is combined with the lysozyme binding peptide. This will result in a novel synthetic construct for specific, non-chromatographic downstream protein purification. Shur, O. et al. 2013, BioTechniques. 54(4): 197-206. Bulutoglu, B. et al, 2017, Submitted.

Engineering the calcium-regulated β-roll peptide for bioseparations applications

253rd National Meeting (2017)

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