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Typically, chromatography is the most costly and time-consuming step in protein purification. As a result, alternative methods have been sought for bioseparations, including the use of stimulus-responsive tags that can reversibly precipitate out of solution in response to the appropriate stimulus. While effective, stimulus-responsive tags tend to require temperature changes or relatively harsh buffer conditions to induce precipitation. Here we describe a synthetic peptide, based on the natural repeat-in-toxin (RTX) domain that undergoes gentler calcium-responsive, reversible precipitation. When coupled to the maltose binding protein (MBP), our calcium-responsive tag efficiently purified the fusion protein. Furthermore, when the MBP was appended to green fluorescent protein (GFP), β-lactamase, or a thermostable alcohol dehydrogenase (AdhD), these constructs could also be purified by calcium-induced precipitation. Finally, protease cleavage of the precipitating tag enables the recovery of pure and active target protein by cycling precipitation before and after cleavage.

The RTX domains found in some pathogenic proteins encode repetitive peptide sequences that reversibly bind calcium and fold into the unique the β-roll secondary structure. Several of these domains have been studied in isolation, yielding key insights into their structure/function relationships. These domains are increasingly being used in protein engineering applications, where the calcium-induced control over structure can be exploited to gain new functions. Here we review recent advances in the use of RTX domains in the creation of calcium responsive biomaterials.