Basic Protein Purification

This protocol has been used for expression & purification of Flu protein (1133 pET28b-M1wt Influenza A virus (A/WSN/1933(H1N1)) segment 7)

Sequence:

MHHHHHHSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILS
PLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAK
EIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMVTTTNPLI
RHENRMVLASTTAKAMEQMAGSSEQAAEAMDIASQARQMVQAMRTVGTHPSSSAGLKDDL
LENLQAYQKRMGVQMQRFKLEHHHHH

PI: 9.15. 29.6 kDa.

1. Grow up Nico21(DE3)/pLysN cells (transformed with 1133 pET28b-M1wt)  in 100 ~150 ml of LB medium supplemented with proper antibiotics (LB/K/C) at 37 oC o/n.

2. Next morning, add 20 ml of culture to 1 L LB medium supplemented with proper antibiotics (K/C), grow at 37 oC.

3. Check OD600, add 0.25  (final concentration) of IPTG when OD600 ~=0.6 to 1.2 (about 2 to 3 hours after culturing in 1 L flask). Induce at 37 oC for 6 hours.

5. Spin down cells, 6000 rpm for 5 min.

6. Transfer to 50 ml tubes using dH2O, spin down cells.

7. Add ~30 ml (per 10 g wet pellet) lysis buffer (Buffer A containing 10% glycerol and 5 mM beta-ME). Freeze cells with dry ice.

8. Thaw cells in 25 oC water bucket.

9. Cells will be lysed upon one freeze/thaw cycle.

10. Homogenize to break sticky DNA.
11. Spin at 12000 RPM for 20 min.

12. Collect supernatant. Do not discard pellet.

13. Spin for 20 min at 25000 RPM (ultracentrifuge), 4 oC.

14. Load cleared supernatant to 10 to 15 ml ml (CV) Ni column, do not throw away flow through (FT).

15. Wash beads with Buffer A until no protein is detectable (using Braford reagent).

16. Wash beads with 20 ml Buffer C (Buffer A with 20 mM immidozol).

17. Elute protein with ~25 to 45 ml Buffer B, collect 3 ml fractions.

18. Measure protein concentration using 10 ul from each fraction and 75 ul Braford reagent.

19.  Combine fractions containing protein.

Dilute 4 volumes with Buffer D

20.  Load diluted sample to 10 ml SP Sepharose XK column preequilibrated with Buffer D.

21. Using FPLC, elute protein with linear gradient from Buffer D to Buffer E (20 CV, 200 ml), collect 3 ml fractions.

22. Combine fractions containing protein.

23. Load to Tosho gel filtration column (FPLC). Separate protein using Buffer G.

24. Combine fractions containing desired protein.

25. Concentrate protein using spin concentators (10 KDa).

26. Measure protein concentration using nanodrop.

27. Add DTT to final conc= 2 mM. Aliquot 1 ml/tube, freeze in dry ice and store them at -80 oC

Buffers:

Buffer A: 10 mM Imidozale, 20 mM Tris-HCl pH 7.0, 600 mM NaCl, 10% Glycerol (optional), 0.3 mM EDTA, 5 mM beta-ME

Buffer B: 300 mM Imidozale, 20 mM Tris-HCl pH 7.0, 600 mM NaCl, 0.3 mM EDTA, 10% Glycerol, 5 mM beta-ME

Buffer C: 20 mM Imidozale, 20 mM Tris-HCl pH 7.0, 600 mM NaCl, 0.3 mM EDTA, 10% Glycerol, 5 mM beta-ME

Buffer D: 20 mM Sodium Acetate Buffer, pH 3.8, mix 12 ml of 0.2M NaOAc and 88 ml of 0.2M HOAc , 5 mM beta-ME, add water  to 1 L, filter through 0.45 u filter.

Buffer E: 20 mM Sodium Acetate Buffer, pH 4.0, mix 18 ml of 0.2M NaOAc and 82 ml of 0.2M HOAc , add solid NaCl to 1 M. Add water to 1 L, filter through 0.45 u filter.

Buffer F: 20 mM Sodium Acetate Buffer, pH 4.0, mix 18 ml of 0.2M NaOAc and 82 ml of 0.2M HOAc , add solid NaCl to 150 mM,  5 mM beta-ME, add water to 1 L, filter through 0.45 u filter.

Buffer G: 20 mM Phosphate buffer, 0.2 M NaCl, 5 mM beta-ME, pH 4.0