Expression and purification protocol of ASK1 (residues 88-973, consisting of TBD, CRR, and KD)

DNA encoding the TBD, CRR and KD domains of human ASK1 (residues 88-973) was cloned into pST39 using XbaI and BamHI sites. 

1. The construct was expressed as a C-terminal 6xHis-tagged protein in Escherichia coli BL21(DE3) cells. The cells were grown at 37°C until an OD₆₀₀ of 0.8 – 1.0 was reached. After that, the bacterial culture was cooled down in a cold room at 4°C for one hour. After that, expression was initiated by induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM. ASK1 TBD-CRR-KD expression was performed overnight at 18 °C.

2. The bacterial culture was centrifuged (20 min at 2,073 g), the pellets were resuspended in lysis buffer (1 x PBS, 1 M NaCl, 4 mM β-ME, and 2 mM imidazole), and the lysate was frozen. 

3. After thawing, the cells were disrupted by incubation with lysozyme and sonication in the presence of phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM. The sonicate was then centrifuged (45 min at 19,561 g and 4 °C) and the supernatant was used to purify the protein.

4. The first purification step was performed using immobilized metal affinity chromatography (IMAC) with a standard protocol. The wash buffer contained 1x PBS, 0.5M NaCl, 2mM β-ME, and 40-60 mM imidazole. The protein was eluted from the column with a buffer consisting of 1x PBS, 0.5M NaCl, 2mM β-ME, and 400 mM imidazole.

5. IMAC was followed by an overnight protein dialysis to remove imidazole. The dialysis buffer contained 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM EDTA, 2 mM β-ME and 10% (w/v) glycerol.

6. After dialysis, the protein was concentrated, and size-exclusion chromatography (SEC) was immediately performed as the second and final purification step on a HiLoad 26/600 Superdex 200 pg column in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol (DTT) and 10% (w/v) glycerol.