Poster abstracts

Poster number 110 submitted by Sabat Gonzalez-Serrano

Rationale protein engineering approach to accelerate a self-removing purification tag

Sabat Gonzalez-Serrano (OSBP), Will Caines (Department of Chemical and Biomolecular Engineering), Dominic Cirillo (Department of Chemical and Biomolecular Engineering)

Abstract:
Recombinant affinity tags are extensively used to purify therapeutic proteins and other biologics; however, one major drawback in bioprocess development is that these fusion tags can become an immunogenicity threat if left on the target proteins (TP). Therefore, any therapeutic that relies on tag-based platforms will require a tag removal step, leading to increased costs and process complexity. Herein, a modified intein, small intervening enzymes that catalyze their excision and the concomitant ligation of flanking polypeptides, was used to mitigate this issue. Our lab has pioneered a protein affinity purification system by modifying the Nostoc punctiforme DnaE split-intein (Npu) into a self-cleaving tag. In this method, the binding of split-inteins mediate the affinity capture of the TP; then a mildly acidic pH (6.2) shift triggers the intein’s self-cleavage, and finally, the tagless product is eluted from a single chromatography column. Although the activation of the Npu system is controlled by its binding assembly and pH, it exhibits variability in self-cleavage rates with different TPs. Previous work has shown that the first two TP residues (+1, +2) can also influence the rate of cleavage. While some TP’s N-termini sequences can be modified to optimize cleavage, other TPs are dependent on conserved native sequences and cannot be altered, thus presenting a challenge for intein-mediated purification methods. Here a rational design approach was used to engineer the Npu intein to achieve faster cleavage with greater tolerance to +1, +2 residues that typically display slower cleavage. This new variant not only shows consistent improvement but also preserves pH controllability as minimal TP cleavage was observed within the first five hours of the reaction at pH 8.5. Advancing this intein-based technology is a key step towards addressing some of the barriers in downstream processing, and will potentially help to expedite the investigation and manufacture of novel therapeutics.

Keywords: Recombiant protein purification, self-removing fusion tags