RedB Purification

Protocol 1

1. Grow pBD24-RedB in BL21-AI (AI: Arabinose inducible) at 37 °C.
2. Dilute overnight culture 1/50 into 1 liter of LB-Amp, grown to an OD600 value of 0.5.
3. Induce with 0.2% (w/v final concentration) of L-Arabinose for 4 hours of induction.
4. Spin down cells, resuspend in 35 ml of resuspension buffer (50 mM Tris-HCl pH 7.5, 10% sucrose).
5. Store cell pellet at -80 °C.
6. Thaw frozen cells at room temperature.
7. Sonicate or homogenize lysate.
8. Spin at 12000 rpm for 20 min.
9. Collectsupernatant, add polyethylenemine dropwise to a 0.5% (v/v) final concentration while stirring. Stir for another 30 min.
10. Spin at 35,000 rpm for 15 minutes at 4 °C. Discard supernatant.
11. Extract pellet with extraction buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 500 mM NaCl, 1 mM DTT, 10% Glycerol).
12. Spin the solution at 12,000 g for 45 minutes.
13. Collect supernatant, add solid ammonium sulphate slowly to 45% (w/v) final concentration while stirring. Let it sit overnight in a cold room.
14. Spin at 12,000 g for 15 minutes.
15. Resuspend the pellet in 35 ml nickel-column BufferA (200 mM NaCl, 20 mM Tris-HCl pH 7.6, 5 mM imidazole), spin at 20,000 g for 30 minutes.
16. Load supernatant into a nickel affinity column.
17. Elute with a 5 mM – 500 mM imidazole gradient in BufferB.
18. Add ammonium sulphate slowly to fractions containing Redβ, to 60% (w/v) final concentration, let sit 30 min.
19. Spin 20,000 g for 30 minutes.
20 Resuspend the pellet in 10 ml MonoQ-A buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 1 mM DTT, 5% Glycerol, 75 mM KCl), and dialyze against the same buffer (500 ml) overnight.
21. Spin to remove insoluble material. Load supernatant to a MonoQ column,
22. Elute with a 75 mM-450 mM gradient of KCl.
23. Dialyse fractions with storage buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 1 mM DTT, 50% Glycerol) overnight at 4 °C.