FIGURE 9.

This figure was adapted from a patent filed by scientists at the Ohio State University (Wood and Shi, 2018; Patent No.: US 10066027 B2; prioritized in 2015, filed in 2016 and approved in 2018, with continuation Patent No.: 10669351 B2) and shows a scheme of split intein immobilization and the corresponding purification method. The N-terminal intein segment (Npu_N) is expressed and purified from a recombinant protein expression host (in this case E. coli), and then immobilized onto a commercially available immobilization resin (e.g., SulfoLink™ resin). The charged resin can be used to directly capture the intein C-terminal intein segment (Npu_C), which has been fused to a Target Protein (TP). After the feed impurities have been washed away, the intein can be induced to release the target protein (TP) from the column matrix by a pH or temperature shift. The cleaved tag-free target can be directly collected from the column, and the column can be regenerated to remove the cleaved intein C-terminal segment, allowing multiple rounds of purification on a single batch of resin.