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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

LARGE SCALE PREPARATION AND PURIFICATION BY AMMONIUM SULFATE PRECIPITATION OF PROTEINS FROM YEAST WHOLE CELL EXTRACT

Large Scale Preparation and Purification by Ammonium Sulfate Precipitation of Proteins from Yeast Whole Cell Extract
Contributor: The Laboratory of Jasper Rine at the University of California, Berkeley
 
Overview
With this protocol, a whole cell extract is prepared from a yeast strain of choice. The cells are lysed using the mechanical force of agitation with glass beads and proteins are partially purified by an Ammonium Sulfate precipitation step. You should determine the behavior of the protein of interest during the salt precipitation before using this protocol by performing a scaled down version of this procedure as some proteins will not be precipitated with the addition of the Ammonium Sulfate.
 
Procedure
1. Grow a 20 liter culture of yeast cells in a fermentor to an Optical Density at 600 nm (OD600) of approximately 5. Measure a 1:10 dilution of the culture for accurate readings in the spectrophotometer.

2. Collect the cells by centrifugation. They may be stored frozen at -80°C at this point.

3. Weigh out approximately 400 g of cells (frozen or thawed).

4. Transfer the frozen cells to a 2 liter beaker and add 400 ml of 2X H Buffer with KCl.

5. To speed the thawing of the cells, place the 2 liter beaker in a 4 liter beaker with about 1.5 liter of room temperature water. Keep the warm water in the 4 liter beaker stirring with a large stir bar on a magnetic stirrer.

6. Move the bead beater apparatus (BioSpec Products) into a cold room and add acid-washed glass beads to within 4 cm of the top of the bead beater (about one-half the volume of the chamber, 200 ml). Remove any excess liquid with a Pasteur pipette.

7. Prepare an ice/H2O/NaCl bath in an ice bucket to chill the chamber during the bead beating. Add NaCl to the ice water until the temperature of the bath is approximately -8°C.

8. After the cells have thawed, add the following to the yeast slurry:

500 mg DTT

1.6 ml of 1 mg/ml Pepstatin A

1.6 ml of 1 M Benzamidine

16 ml of 0.1 M PMSF

9. Add a portion of the cells to the bead beater. (They all won't fit in at once.) Bead beat the cells for 30 sec on and 90 sec off for 20 min (i.e. approximately 10 cycles). It will take about 5 to 6 rounds to do all 400 g of yeast cells. Make sure to keep the ice/H2O/NaCl jacket at approximately -5°C. To accomplish this, change the bath every 10 min.

10. Take a small aliquot of cells (2 μl) after each round of bead beating and check the percent cell breakage under a microscope. Breakage should be at least 75%.

11. Pool the lysates from the first three rounds and pool the lysates from the second two (or three) rounds of breakage. Measure the volumes. Add one-twelfth volume of Saturated Ammonium Sulfate to each pool.

12. Allow the cells to stir gently for 30 min on ice (see Hint #2).

13. Centrifuge the cells for 20 min at 13,000 X g (9,000 rpm using a Sorvall GSA rotor) at 4°C.

14. Transfer the supernatant to six Beckman 45 Ti bottle assemblies (the bottles hold 70 ml each).

15. Centrifuge the bottles at 150,000 X g (44,000 rpm using a 45 Ti rotor) for 90 min (see Hints #3 and #4).

16. Collect the supernatants using a 25 ml pipette. Avoid the cloudy material at the bottom of the tubes (i.e. the last 5 to 10 ml) as it is predominantly DNA that will not be removed in subsequent steps.

17. Measure the volume of the recovered supernatant and add 0.27 g of finely ground powdered solid Ammonium Sulfate per ml of collected supernatant (see Hint #5).

18. Once all the Ammonium Sulfate is in solution, stir the solution on ice for 30 min.

19. Centrifuge to pellet the precipitate at 125,000 X g (40,000 rpm using a 45 Ti rotor) for 30 min.

20. Pour off the supernatant and drain the pellet by inverting the tube over lab tissue.

21. For a 400 g preparation, resuspend the pellets in approximately 250 ml of Buffer H without KCl (see Hint #6).

22. Dialyze the resulting material against Buffer H with 0.1 M to 0.15 M KCl. (Use 0.1 M KCl for a quick dialysis and 0.15 M KCl for an overnight dialysis.)

23. Measure and record the final volume, conductivity, protein concentration, and total protein amount (see Protocol on Determination of Protein Concentration).

Solutions
1 mg/ml Pepstatin A
Saturated Ammonium Sulfate   to 1 liter of ddH2O
Add 697 g ammonium sulfate
Store at 4°C
pH 7.5
Buffer H with 0.1 M or 0.15 M KCl   0.1 M or 0.15 M KCl (see Protocol Step #22)
5 mM Magnesium Acetate
2 μM Pepstatin A*
0.02% (v/v) Igepal CA-630
50 mM HEPES-KOH, pH 7.5
0.5 mM Phenylmethylsulfonyl Fluoride (PMSF)*
2 mM DTT*
1 mM Benzamide*
1 mM EGTA
1 mM EDTA
*Add right before use.
10% (v/v) Glycerol
Buffer H without KCl   5 mM Magnesium Acetate
2 μM Pepstatin A*
0.02% (v/v) Igepal CA-630
50 mM HEPES-KOH, pH 7.5
0.5 mM Phenylmethylsulfonyl Fluoride (PMSF)*
2 mM DTT*
1 mM Benzamide*
1 mM EGTA
1 mM EDTA
*Add right before use.
10% (v/v) Glycerol
Buffer H (2X) with KCl   0.2 M KCl
0.04% (v/v) Igepal CA-630
1 mM Phenylmethylsulfonyl Fluoride (PMSF)*
2 mM Benzamide*
2 mM EGTA
10 mM Magnesium Acetate
20% (v/v) Glycerol
2 mM EDTA
4 μM Pepstatin A*
*Add right before use.
4 mM DTT*
100 mM HEPES-KOH, pH 7.5
0.1 M PMSF   Prepare in DMSO (CAUTION! see Hint #1)
1 M Benzamidine
 
BioReagents and Chemicals
Pepstatin A
Benzamidine
Potassium Chloride
Sodium Chloride
Ammonium Sulfate
Glass Beads, Acid Washed
PMSF
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Vigorous stirring will result in the increased breakage of DNA at this stage, which will prevent it from precipitating in the subsequent ultracentrifugation.

3. The use of a Beckman ultracentrifuge is crucial because other centrifuges take too long to bring the 45 Ti rotor up to 44,000 rpm and take too long to slow it down at the end of the centrifugation.

4. Make sure that the 45 Ti bottle assemblies are filled to prevent bottle failure at this high RCF.

5. Use a coffee grinder (dedicated to chemical use) to grind the solid Ammonium Sulfate and add the solid slowly as the solution is stirring on ice. Adding it all at once will cause precipitation of unwanted protein due to high local concentrations of the undissolved salt.

6. Use a large glass homogenizer and 1 ml pipettor for a fast resuspension.