Fig. 2

Comparison of previously reported SPI fusion protein isolation protocols with the new protocol outlined in this work. (A) Previously reported isolation protocols to obtain pure peptide from a SPI fusion protein (Lamer et al., 2022a). Clarified cell lysate is passed over a Ni-NTA column, and the SPI fusion protein is eluted with imidazole (purple hexagons). The intein is then cleaved via incubation in 100 mM DTT overnight, and then removed from the sample with a chitin column (top arrow). DTT and imidazole are removed with gel filtration chromatography prior to addition of the SUMO protease. The cleaved His₆-SUMO tag and His-tagged SUMO protease are removed with a second Ni-NTA column, and the peptide in the column flow through is finally purified with HPLC. Alternatively, if the use of chitin resin is undesirable (bottom arrow), after intein cleavage the buffer is exchanged with gel filtration to remove imidazole and DTT, and the His₆-SUMO-peptide protein is separated from the intein using a second Ni-NTA column. Imidazole is then removed from the eluted protein sample with dialysis, and then cleavage by the SUMO protease, a third Ni-NTA column, and HPLC are conducted to obtain pure peptide. (B) Simplified isolation protocol used in this work to obtain pure peptide from a SPI fusion protein. The SPI fusion protein is isolated from the clarified cell lysate using a Ni-NTA column, and then the intein is cleaved from the eluted SPI protein by addition of 100 mM βME. Reducing agents and imidazole are then removed with dialysis, and addition of the SUMO protease cleaves the His₆-SUMO tag from the peptide. The peptide is then isolated using a second Ni-NTA column, and finally purified with HPLC.