pASK-TS-MCoTI

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	E. coli Origami 2(DE3) cells (Novagen) were transformed with plasmids pASK-SFTI-C3 or pASK-SFTI-C11. Expression was carried out in 1 L of 2XYT or M9 media containing ampicillin (100 μg/L) at room temperature. Briefly, 5 mL of an overnight starter culture were used to inoculate 1 L of 2XYT or M9 media. Cells were grown to an OD at 600 nm of ≈ 0.6 at 37° C. Protein expression was induced by addition of anhydrotetracycline hydrochloride (AHT) to a final concentration of 200 μg/L at room temperature for overnight.

Protocol tips

E. coli Origami 2(DE3) cells (Novagen) were transformed with plasmids pASK-SFTI-C3 or pASK-SFTI-C11. Expression was carried out in 1 L of 2XYT or M9 media containing ampicillin (100 μg/L) at room temperature. Briefly, 5 mL of an overnight starter culture were used to inoculate 1 L of 2XYT or M9 media. Cells were grown to an OD at 600 nm of ≈ 0.6 at 37° C. Protein expression was induced by addition of anhydrotetracycline hydrochloride (AHT) to a final concentration of 200 μg/L at room temperature for overnight.

Publication protocol

E. coli Origami 2(DE3) cells (Novagen) were transformed with plasmids pASK-SFTI-C3 or pASK-SFTI-C11. Expression was carried out in 1 L of 2XYT or M9 media containing ampicillin (100 μg/L) at room temperature. Briefly, 5 mL of an overnight starter culture were used to inoculate 1 L of 2XYT or M9 media. Cells were grown to an OD at 600 nm of ≈ 0.6 at 37° C. Protein expression was induced by addition of anhydrotetracycline hydrochloride (AHT) to a final concentration of 200 μg/L at room temperature for overnight. The cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl at pH 8.0 containing 5% glycerol) and then lysed by sonication. The lysate was clarified by centrifugation at 15,000 rpm in a Sorval SS-34 rotor for 30 min. The clarified supernatant was incubated with 1 mL Ni-NTA agarose beads (Qiagen), previously equilibrated with lysis buffer at 4° C for 1 h with gentle rocking. The beads were separated from the cell lysate by centrifugation and then extensively washed with 50 bed-volumes of wash buffer (20mM imidazole, 50 mM NaH2PO4, 300 mM NaCl buffer at pH 8.0). Quantification of the level of expression was performed by quantifying the IC and IN polypeptides by SDS-PAGE analysis (Fig. 4A) was performed to quantify the expression level of intein. The expression level for intein precursor 1a and 1b was estimated to be ≈110 mg/L and 135 mg/L, respectively.

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