Current affinity tag-based protein purification approaches are not ideal for biotechnological applications. We report the development of a column-free, protease-free protein purification technology that takes advantage of an engineered split-intein and a stimulus-responsive elastin-like-peptide (ELP) tag. Under reducing condition, this engineered split-intein catalyzes trans-splicing reaction pH 6 and a single C-terminal cleavage reaction at pH 8. The ELP is fused to the N-terminal fragment of the split intein and the protein of interest (POI) to the C-terminal fragment of the intein. These two fusion proteins are expressed individually in E. coli and mixed after cell lysis. Under low salt non-reducing conditions, the N- and C-inteins associate strongly with each other without cleavage, physically connecting the ELP to POI. High salt conditions trigger ELP-mediated phase separation of the POI. Next, the precipitate containing the POI is resuspended in a low-salt, reducing buffer to resolubilize the aggregate and induce intein cleavage, releasing the POI from the ELP-intein complex. Finally, the resulting mixture is subjected to high salt conditions again to precipitate out the undesired ELP-intein complex from the solution, leaving only purified POI in the solution phase. This protein purification technology is simple, convenient and rapid. The entire process from soluble cell lysate to purified tag-free protein takes <4h. This technology should facilitate cost-effective purification of proteins on a large scale.

Column-free, protease-free protein purification via mini-intein cleavage

243rd National Meeting (2012)

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