Table 1.
Dialysis-based procedure for an IDP sample from inclusion bodies using the Npro (EDDIE) fusion protein expression system
| 1. Pick 3 to 5 colonies of E. coli BL21 (DE3) and add them to 1 mL LB medium, and grow the culture overnight at 37°C. |
| 2. Add 0.5 mL of the bacterial culture to 100 mL of the medium (either M9 medium containing 15NH4Cl or LB depending on the planned NMR experiments) and incubate at 37°C for 12–16 h (pre-culture medium). |
| 3. Add the pre-culture medium to 900 mL medium and incubate at 37°C until the OD (600 nm) of the culture is 0.6–0.7 (main culture medium). |
| 4. Induce protein expression with the addition of 1.0 mM IPTG (final concentration). |
| 5. Incubate the culture at 37°C for an additional 4 h. |
| 6. Harvest the cells. |
| 7. Disrupt the cells by sonication in the buffer containing 20 mM sodium-phosphate buffer (pH 8), 75 or 300 mM NaCl, 5 mM EDTA (sonication buffer). The buffer volume is 10–20 mL per 1 g wet E. coli cells. |
| 8. Centrifuge and collect the pellet fraction (IBs). |
| 9. Wash IBs with the sonication buffer supplemented with 0.1% Triton-X100 twice. |
| 10. Solubilize IBs with buffer containing 50 mM Tris-HCl (pH 7.5), 8 M urea, 300 mM NaCl and 25 mM DTT. Incubate the solution at 4°C overnight. |
| 11. Dilute the volume 5 times with the same buffer without DTT. The final concentration of DTT is now 5 mM. |
| 12. Apply the Npro fusion proteins to an immobilized Ni2+ affinity column His-AcceptTM. |
| 13. Wash the resin. |
| 14. Elute the sample with increasing concentrations (100, 200, 300, 400, and 500mM) of imidazole. |
| 15. Dialyze the protein sample against the refolding buffer (1 M Tris-HCl, pH 7.5, 5% glycerol, 10 mM DTT and 2 mM EDTA. Repeat this step twice. At the second step, keep sample at room temperature and allow autocleavage for another 16 h. |
| 16. Dialyze the protein sample against the precipitation buffer containing 25 mM Tris-HCl, pH 7.5 (at 4°C for 16 h). |
| 17. Purify the peptide by RP-HPLC. |