A Designed, Phase Changing RTX-Based Peptide for Efficient Bioseparations

Abstract

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.

Recombinant protein expression in Escherichia coli: advances and challenges

“…The addition of β roll tags to a recombinant protein allows for its selective precipitation in the presence of calcium. The final products presented a high purity and the precipitation protocol only takes a couple of minutes (Shur et al, 2013). Another protein-based stimulus-responsive purification tags are elastin-like polypeptides (ELPs), which consist of tandem repeats of the sequence VPGXG, where X is Val, Ala, or Gly in a 5:2:3 ratio (Meyer and Chilkoti, 1999).…”

Aggregating tags for column‐free protein purification

“…A second protein, repeat in toxin (RTX), a calcium‐responsive polypeptide, is also used in a similar fashion. This protein consists of tandem repeated nonapeptide sequences GGXGXDXLX (from five to more than 50 repeats), where X is any amino acid and L can be replaced by I, F, V, or Y occasionally [9, 60]. RTX has been found to switch from a disordered structure to a parallel β;‐roll structure in the presence of calcium ions [60].…”

“…RTX has been found to switch from a disordered structure to a parallel β;‐roll structure in the presence of calcium ions [60]. Chenal et al identified the universal sequence GGAGNDTLY from a RTX‐containing protein database [9]. This group then devised a purification and separation strategy similar to the one used for Annexin B1 fusion expression, which employs a RTX tag of 153 residues (17 repeats) and an enterokinase (EK) cleavage site.…”

“…MBP, GFP, β;‐lactamase and alcohol dehydrogenase (AdhD) were successfully purified with this strategy. At the shake flask level, the yields of the fusion proteins were reported to be in excess of 176 mg/L, but the final yields for the target proteins were not reported (Table 1) [9].…”

“…In recent years, a new class of aggregating tags has been exploited in column‐free isolation of proteins and peptides [7–10]. These tags contain a protein or peptide that induces the fusion protein to form aggregates (during expression or after expression) and a cleavage site or a cleavage tag that is used to release the target protein from the fusion protein under suitable conditions (chemical cleavage, protease cleavage, self‐cleavage mediated by inteins).…”