Chapter 4:

Section 4.1
Making the Crude Extract

Background

The first step in protein purification is to make a cell-free extract from the source material so that the target protein is in solution. The source material consists of any biological material that contains an abundant source of the target protein. Source material may be a culture of microorganisms, cells grown in culture, plants, or tissues isolated from an animal, such as liver, lung, ovary, muscle, brain. When using cultured cells or microorganisms, if the target protein is secreted outside the cell, no cell disruption step is needed-- it is only necessary to spin down the cells and harvest the supernatant. However, most proteins are located inside the cell and must be released into solution by some type of disruption process. Disruption procedures vary from gentle (osmotic lysis) to harsh (sonication) depending on the cell type or tissue type that is the source of your target protein (Table 4.1). For example, making an extract from red blood cells requires only a mild disruption step while extracting plant tissue requires a much harsher process in order to break down the tough cell walls of the plant.

ß-galactosidase is an intracellular protein made by E. coli. Like many bacteria, E. coli is encased in a tough cell wall to protect it from a potentially hostile environment. In this procedure, you will be using a method called sonication to break open the bacterial cells. Sonication damages the cell wall by high frequency sound waves. The cells are disrupted in a buffer that has been chosen to keep the target protein in an active form. After the cell disruption step, cells and other insoluble debris are separated from the extract by centrifugation. If the cells have not been completely disrupted, much of the protein will not be released into solution, and will be lost with the cell debris.

Extraction Buffer: The composition of the extraction buffer is important for maintaining structure and function of the target protein and is based on the the unique characteristics of the target protein. The buffering pH is based on the pH stability range of the protein. Other components such as ionic strength, divalent cations (Ca⁺⁺ and Mg⁺⁺), or reducing agents (dithiothreitol or ß-mercaptoethanol) may be needed to maintain activity. In making the extract, cells are lysed and proteases (enzymes that degrade proteins) are released from their intracellular compartments. These enzymes may damage the target protein. To prevent proteases from digesting the target protein, two strategies are commonly followed:

• The extract is kept cold. The activity of proteolytic enzymes is greatly reduced by cold temperatures. For this reason, the protein purification process is often conducted in cold rooms. At the very least, an effort is made to keep the extract at 4°C.
• Protease inhibitors are sometimes added to the mixture to prevent degradation by proteases. The drawback to this strategy is that the inhibitors must eventually be removed, along with other contaminant proteins.

The extraction buffer for this ß-galactosidase purification is called Breaking Buffer and the recipe is found in Appendix A. The dithiothreitol (DTT) should be added just before use since it breaks down over time. Sonication generates a lot of heat and it will be necessary to use an ice bath with ethanol to preserve activity of the target protein.

Procedure 4.1

1. Examine the sonicator. Find the probe of the sonicator within the soundproof box. Find the ring stand, clamps, etc that will be used to position the sample in the chamber of the sonicator.
2. You will receive a sample of frozen E. coli weighing approximately 8 grams. The exact weight should be marked on the tube. Thaw the cells on ice.
   Note: Usually, 100 mLs of culture will yield about a gram of wet cell paste.
3. While the cells are thawing, choose a container for your sample. The container should be small enough so that the sonicator probe is immersed deeply in the sample but large enough so that the probe does not touch the sides or bottom of the container. (50 mL glass tubes with round bottoms work well.)
4. Resuspend your thawed cells in breaking buffer. The necessary volume (in ml) of breaking buffer is determined by multiplying the weight of the cells (in grams) by a factor of five. (Remember that you are expected to add dithiothreitol (DTT) to the breaking buffer. Check the recipe in Appendix A to find out how much to add.) So if you have 8 grams of cells you will need 40 (8 x 5) mL of breaking buffer to resuspend the cells. The cell paste is viscous and a rubber policeman will probably be necessary – gentle vortexing is permitted at this stage only.
5. Prepare an ethanol ice bath in a beaker and place the beaker securely on a ring stand in the sound proof box of the sonicator.
6. Arrange everything so that the probe will be prevented from touching the sides of the glass while operating. It is useful to move the tube up so that the probe touches the bottom and then back off so that the probe does not touch the bottom. Make sure everything is clamped securely to prevent any slippage.
7. Lyse your cells with the sonicator using a 50% power setting for 12 minutes. We are using a Branson Sonifier 250. For other models follow the manufacturer’s recommendations.
8. Pre-cool the centrifuge and rotor that you will be using to 4°C. Determine the appropriate rotor speed to obtain 10,000xg in the rotor.
9. Clean thoroughly a centrifuge bottle or tube such as the 250 ml polycarbonate centrifuge bottle (hard clear plastic). Pour your sample into this bottle and prepare another bottle for balance or balance with another group’s sample. Do not use polystyrene or polypropylene tubes (blue cap conicals) at these speeds.
10. Spin out the cell debris at 4°C for 30 minutes at 10,000 xg. (Don’t forget to fill out the centrifuge log book.)
11. Remove the supernatant. (You may use a pipette or pour off the supernatant gently into a clean container.) This supernatant is your crude extract. Save the pellet until enzyme assay verifies that enzyme activity is present in the extract. Record the volume of the extract and save 3 ml in approximately 0.5 ml aliquots for assays. Store the aliquots in the refrigerator. Remember to write in your lab notebook where the aliquots are stored. Store the crude extract at 4°C – the entire volume of crude extract (except for the aliquots) will be used for the next purification step, ammonium sulfate precipitation.
12. Optional: Compare the effect of storage temperature on enzyme activity over time. Try storing one aliquot in the refrigerator and one in the freezer to see whether you enzyme is more stable over time at 4°C or -20°C.

Assays

• At this point, you will need to determine the enzyme activity of the crude extract according to the enzyme assay procedure outlined in Section 3.2. In the enzyme assay procedure, recall that you are adding 10-50µl of sample to 1 ml of Z buffer. After the sonication and centrifugation step, your sample is in breaking buffer so dilutions should be made in breaking buffer before removing an aliquot of 10-50µl and adding it to the Z buffer. What will be the composition of your blank tube?
• Once you have determined that you have enzyme activity in your extract, proceed with the next step in the purification, ammonium sulfate precipitation, Procedure 4.2.
• As time permits, determine the protein concentration and specific activity according to the assay methods in Chapter 3.

Alternate Extraction Method

Procedure 4.1b; BugBuster™ reagent from Novagen,

1. You will receive a sample of frozen E. coli weighing approximately 8 grams. The exact weight should be marked on the tube. Thaw the cells on ice.
2. Verify that Dithiothreitol (DTT) has been added to the BugBuster™ buffer to a final concentration of 5 mM.
3. Resuspend the cell paste in room temperature BugBuster reagent using 5 ml reagent for each gram of wet cell paste. DO NOT ADD this volume all at once. The cell paste is viscous and a rubber policeman will probably be necessary – gentle vortexing is permitted if necessary at this stage only.
   
   Recommended method: Add a couple mLs of buffer to the cell paste and gently rock the tube back and forth (tapping is OK), work with a rubber policeman as needed. Repeat the process with another ml or two of buffer each time until you have suspended all the cell paste (or MOST of it). It is a good idea to avoid frothing and bubbles. (Why?)
4. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10-20 minutes at room temperature.
5. Pre-cool the centrifuge and rotor that you will be using to 4°C. Determine the appropriate rotor speed to obtain 16,000xg in the rotor. (check Appendix D for a centrifugation chart)
6. Clean thoroughly a centrifuge bottle or tube. Pour your sample into this bottle and prepare another bottle for balance or balance with another group’s sample. Do not use polystyrene or polypropylene tubes (blue cap conicals) at these speeds.
7. Spin out the cell debris at 4°C for 20 minutes at 16,000 xg. (Don’t forget to fill out the centrifuge log book.) (10,000xg for 30 minutes will also work)
8. Remove the supernatant. (You may use a pipette or pour off the supernatant gently into a clean container.) This supernatant is your crude extract. Record the volume of the extract and save 0.5 ml in approximately 0.1 ml aliquots for assays. Store the aliquots in the refrigerator. Remember to write in your lab notebook where the aliquots are stored. Store the crude extract at 4°C – the entire volume of crude extract (except for the aliquots) will be used for the next purification step, ammonium sulfate precipitation.
   
   Although you expect most of the activity to be in the supernatant, resuspend the pellet and save it for later enzyme assay to compare to the extract.

Alternate Extraction Method: Procedure 4.1c

Acceptable disruption can also be achieved using a mortar and pestle and an abrasive, inert material such as glass beads or alumina. Alumina (aluminum oxide) is a fine white material similar in appearance to salt.

1. Freeze the E. coli cell pellets on parafilm (about 2 gms). After an hour at
   -20°C, place the cells in a mortar and pestle (prechilled) and grind them with 2-3 times the cell weight of alumina (Sigma #) for 20 minutes.
2. Add 5 cell volumes of breaking buffer and continue to work with the mortar and pestle for about 5 minutes. The volume (in ml) of breaking buffer is determined by multiplying the weight of the cells (in grams) by a factor of five. (Remember that you are expected to add dithiothreitol (DTT) to the breaking buffer. Check the recipe in Appendix XY to find out how much to add.)
3. Pour the slurry into a centrifuge tube and spin out the glass beads and cell debris for 20 minutes at 10,000 xg in a refrigerated centrifuge. (If a superspeed centrifuge is not available, a clinical centrifuge may also be used for 30 minutes at top speed although it will not be as effective.)
4. Remove the supernatant and take aliquots as described in procedure 4.1, step 11.

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