(623x) Non-Chromatographic Protein Purification Via Mini-Intein Cleavage

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 pH-sensitive split-intein and a stimulus-responsive elastin-like-polypeptide (ELP) tag. This engineered split-intein catalyzes a single C-terminal cleavage reaction in the presence of reducing agent. 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. In the absence of reducing agent, 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 highly efficient and should facilitate cost-effective purification of proteins on a large scale.