(280b) A Streamlined Protein Purification Platform for Toxic Proteins Based on Intein Trans-Splicing

Shakalli Tang, M., The Ohio State University
Shi, C., Ohio State University
Wood, D. W., The Ohio State University

Many important functional proteins often exhibit toxicity when overexpressed in heterologous hosts.  Unfortunately, this toxicity can complicate the production of these proteins in recombinant systems, which can slow their characterization.  Although a number of engineered expression strains and plasmids have been developed to optimize toxic protein expression, many targets remain recalcitrant in these systems due to their toxicity to the expression host.  In this work, we have developed a novel protein purification platform based on intein trans-splicing, with special relevance for proteins that are extremely toxic to recombinant host cells.  In this system, the toxic protein is split into two inactive fragments, which are each separately expressed in fusion to the segments of a split intein.  To purify the active product protein, the N-terminal intein segment is first immobilized onto an affinity column via a fused affinity tag.  The C-terminal intein segment is then added to the column, where it specifically associates with the immobilized segment.  The assembled intein segments then splice to deliver the mature target protein, simultaneously releasing the purified target from the affinity column.  To demonstrate this method, we generated a hybrid DnaE intein consisting of the N-terminus of the Npu DnaE intein and the C-terminus of the Ssp DnaE intein. The hybrid intein exhibits tolerance for a wider range of amino acids at the +2 site of the C-terminal splicing junction than has been observed with the Npu intein alone.  In the production of the highly toxic homing endonuclease I-TevI, the yield from the hybrid intein is 20% higher than the native Npu DnaE intein, while the I-TevI protein purified from both inteins showed native activity.