Prepare-Cel1-from-Celery

Protocol 1

Note:
• Have ready all PMSF and Tris-Buffer solutions
• Thaw aliquots of PMSF solution immediately before use
• Use a sieve to remove floating debris

1.  Prepare celery for juicing:
    a. Cut off the bottom from each bunch to separate stalks
    b. Trim off tops, leaves, and tough, white bottoms from each stalk
    c. Rinse with water and dry with paper towels
2.  Blender celery at 4° C to obtain 1.5 L juice
3.  Make a 0.1 M Tris-HCL, pH 7.7, 100 ÎŒM PMSF solution in celery juice
     a. per L juice add: 50 ml 2M Tris pH 7.7
     b. per L juice add: 1 ml 0.1 M PMSF
4.  Centrifuge the juice solution at 15,000 g for 20 minutes, 4 °C
5.  Decant and save supernatant, discard pellet
     a. Pellet: fibrous and sticky, bright green
     b. Supernatant: cloudy greenish-yellow
6.  Bring supernatant to 25% ammonium sulfate
     a. per L supernatant add: 144 g (NH4)2SO4
     b. Volume will increase marginally (3%)
7.  Mix solution gently for 1 hour, 4 °C
8.  Centrifuge solution at 15,000 g for 45 minutes, 4 °C
9.  Decant and save supernatant, discard pellet
     a. Pellet: small and sticky, green or light gray
     b. Supernatant: cloudy brownish-yellow
10.  Bring supernatant to 80% ammonium sulfate
     a. Per L supternatant add: 390 g (NH4)2SO4
     b. Volume will increase significantly (25%)
11.  Mix solution gently for 1 hour or overnight, 4 °C
12.  Centrifuge solution at 15,000 g for 90 minutes, 4 °C
13.  Save pellet, decant and discard supernatant
       a. Pellet: large compared to previous pellets, yellowish-brown or beige
       b. Supernatant: transparent brownish-yellow
14.  Store protein-precipitate pellet at 4 °C (weeks) or -80 °C (indefinite)

Dialysis

Caution:
• Use Gloves in All Steps, Note that PMSF is TOXIC.
• PMSF will recrystallize at 4 °C – thaw and use immediately
• PMSF will degrade over time in aqueous solutions – do not allow to sit in solution

Prepare 5 L Buffer B: 

a. 0.1 M Tris pH 7.7	add 50 ml 2 M stock
b. 0.5 M KCl	add 250 ml 2 M stock
c. 0.01% Triton X-100	add 5 ml 20% stock
d. 100 ÎŒM PMSF	add 5 ml 0.1 M stock

Resuspend pellets in a volume of Buffer B equivalent to 1/10th the initial volume of celery juice
   a. For a 1.5 L prep, resuspend in a 150 ml solution of protein in Buffer B
   b. Scrape pellet from wall of centrifuge bottle using a metal spatula
   c. Smear or break down large chunks of pellets for easier solvation
   d. Vortex gently by hand and avoid over-foaming solution
   e. Transfer buffer and pellets from tube to tube until all pellets are combined
   f. Resuspended pellet should be homogeneous and free of debris

Add suspended protein to dialysis tubes

Dialyze in 5 or 6 changes of 5 L Buffer B
   a. Stir solution slowly to avoid drawing dialysis tubes down into the stir bar
   b. Allow last dialysis to proceed for at least 4 hours or overnight

Collect the dialyzed protein solution and aliquot
   Aliquot into labeled and dated 1.5 or 2.0 ml tubes

Store aliquots at -80 °C

Protocol 2

1. Add 500 ml of 0.1 M TrisHCl, pH 7.7 with 100 µM PMSF to 0.5 kg of store-bought celery.
2. Chop with a blender at 4°C.
3. Spin for 20 min at 6000 rpm to pellet debris.
4. Add (NH₄)₂SO₄ to supernatant to adjust to 25% saturation.
5. Put on ice for 30 min.
6. Spin at 12000 rpm for 30 min at 4°C
7. Add (NH₄)₂SO₄ to the resulting supernatant to 80% saturation.
8. Put on ice for 30 min.
9. Spin at 25000 rpm for 30 min.
10. Resuspend the pellet in 50 ml of 0.1 M Tris-HCl, pH 7.7, 100 mM PMSF (1/10 starting volume).
11. Dialyse the suspension with 10K MW cut-off against a total of 8L of the same buffer, with four changes every 4 h.
12. Store aliquots of extract at -20°C or -80°C

Protocol 3

1. Use a Blender to chop Celery stalks (7 kg) at 4 ^oC.
2. Adjusted with a 10× solution to give the composition of buffer A (0.1 M Tris–HCl, pH 7.7, 10 mM phenylmethanesulfonyl
fluoride (PMSF).
3. Spin for 20 min at 6000 rpm to pellet debris.
4. Add (NH4)2SO4 to supernatant to adjust to 20% saturation.
5. Put on ice for 30 min.
6. Spin at 12000 rpm for 30 min at 4°C
7. Add (NH4)2SO4 to the resulting supernatant to 70% saturation.
8. Put on ice for 30 min.
9. Spin at 25000 rpm for 30 min.

10. Dissolve the final pellet in 250 ml buffer A and dialyze against 0.5 M KCl in buffer A.

11. Incubate the solution with 10 ml concanavalin A–Sepharose resin (Sigma) overnight at 4 oC.

12. Pack the slurry into a 2.5 cm diameter column and wash with 0.5 M KCl in buffer A.

13. Elute bound CEL-I with 90 ml 0.3 M a-D+-mannose, 0.5 M KCl in buffer A at 65 oC.

14. Dialyse CEL-I against buffer B (25 mM potassium phosphate, 10 mM PMSF, pH 7.0).

15. Apply the dialysed solution to a 100 ml phosphocellulose P-11 column that had been equilibrated in buffer B.

16. Elute the bound enzyme with a linear gradient of KCl in buffer B.

17. Pool fractions containing  CEL-I activity.

18. Add ammonium sulfate to saturation, incubate on ice for 30 min.

19. Spin to collect precipitate.

20. Dissolve the precipitate in Buffer C (50 mM Tris–HCl, pH 7.8, 0.2 M KCl, 10 mM PMSF, 1 mM ZnCl₂).

21. Dialyse against Buffer C.

22. Load to a Superose 12 FPLC column equilibrated with Buffer C.

Digestion Buffer and Condition

After PCR gene synthesis, to 50 µL PCR reaction add:

10 µL of 10X digestion buffer (100 mM KCl, 100 mM MgSO₄, 100 mM Tris-HCl, pH 7.5, 0.02% Triton X100, 0.002 mg/ml BSA)

5 µL of Cel-1 Nuclease

35 µL water

Incubate at 45°C for 15 min

Alternative Condition

CEL 1 digestion solution: 10mM HEPES (pH 7.5), 10 mM MgS0₄, 0.002% (w/v) Triton X-100, 20 ng/ml bovine serum albumin, and 1:300-1:10,000 dilution of CEL 1.