PURIFICATION OF BACTERIOPHAGE T7 4A’, 4B, AND
MUTANT PROTEINS
Day 1. TRANSFORMATION: This should be done just one day before the
large-scale growth of cells.
1. Thaw BL21-DE3 competent cells on ice. Add about 0.1 g of the plasmid to
the competent cells and gently mix.
2. Place the cells on ice for 30 minutes.
3. Heat the tubes in a 42 C water bath for exactly 40 seconds. Return the cells
to ice for 2 minutes.
4. Add 200 L of the LB media (room temperature) to the tube containing the
cells, and mix completely.
5. Allow the cells to grow in the 37 C incubator for 1 hour.
6. Spread the transformed cells on a LB-ampicillin agar plate (prewarmed plate
at 37 C).
7. Let the plates incubate upside down at 37 C for 12 to 14 hours (4B takes
longer).
Day 1. Getting ready for GROWTH AND INDUCTION OF CELLS
1. Autoclave 12 two liter flasks containing one liter of LB media and 500 ml of
LB media in a bottle the day before.
2. Aliquot 10 ml of LB in 12 culture tubes.
Day 2. GROWTH AND INDUCTION OF CELLS (This procedure takes about
14-15 hours)
1. Early in the morning (around 7 am), pick a single colony from the plate and
place it in a test-tube containing 10 mL of autoclaved LB media and 1X
ampicillin (100ug/ml).
2. Allow the cells to grow at 37 C in a shaker until you begin to see them in the
culture (it takes about 6 hours).
3. Add one tube of cells to one flask containing one liter of LB with ampicillin
and shake at 37C (prewarm the flasks in the 37C incubator shaker).
4. After about 5 hours take the OD, and at an OD of 0.5-0.6 add IPTG to a final
concentration of 0.5 mM to induce protein expression.
5. Shake for another 3 hours.
6. Spin down the cells by centrifuging at 4000-5000 rpm for 15 minutes.
7. Decant off the media, weigh the cells, and resuspend the cells in no more
than 200 mL of 1X buffer A.
8. Place the resuspended cells in polypropylene tubes (50 mL tubes) and store
them in the -80 C freezer.
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GETTING READY FOR THE PROTEIN PREP (days before the prep).
Preparing Buffers: Three different buffers are used during a prep. Buffer A is
made up as 1X and stored at 4 C. Buffers B and C are made up as 10X buffers
and stored at room temperature.
1X Buffer A: 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10%
sucrose.
To make 1X Buffer A
1. Prepare 500 mL of 0.1 M Tris Cl, pH 7.5.
2. Add NaCL (25 mL of 4 M)
3. Add EDTA (2 mL of 0.5 M)
4. Add DTT (1 mL of 1 M DTT)
5. Add 100 g sucrose
6. Add water to 1 L
1X Buffer B: 20 mM potassium phosphate, pH 7.2, 0.1 mM EDTA, 1 mM DTT,
10 % glycerol.
To make 10X Buffer B:
1. Add 200 mL of 1 M potassium phosphate, pH 7.2 (made from the
monobasic and dibasic salts).
2. Add EDTA ( 2 mL of 0.5 M)
3. Add water to 1 L
To make 1X Buffer B:
1. Add 100 mL of the 10X Buffer B
2. Add 0.154 g DTT
3. Add 100 mL glycerol
4. Add water to 1 L
Note: there is no NaCl added to Buffer B above. You will be using 1X Buffer B
with 50 mM NaCl and 1X Buffer B with 500 mM NaCl. Add the necessary
amount of NaCl as needed.
1X Buffer C: 50 mM Tris, pH 7.5, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol
To make 10X Buffer C:
1. Make 0.5 M TrisCl, pH 7.5 (60.55 g Tris; lower pH to 7.5 with HCl)
2. Add EDTA (2 mL of 0.5 M EDTA)
3. Add water to 1 L.
To make 1X Buffer C:
1. Add 100 mL of the 10X Buffer C
2. Add 0.154 g DTT
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3. Add 100 mL glycerol
4. Add water to 1 L
Note: there is no NaCl added to Buffer C above. You will be using 1X Buffer C
with varying amounts of salt. Add the NaCl as required.
Cycling Phosphocellulose: (a day or two before the prep)
1. Weigh out 25 g of dry phosphocellulose.
2. Add the phosphocellulose to 500 mL of 0.5 M NaOH in a 4-L beaker. Stir the
phosphocellulose gently to avoid creating fines, and let it settle to the bottom
of the beaker. Decant the excess solution after 5 minutes. Do not let the
phosphocellulose sit in the NaOH for more than 5 minutes.
3. Fill the 4-L beaker with several liters of distilled water and again gently stir the
phosphocellulose. Let it completely settle to the bottom (takes about 5
min), and decant off the excess water. Repeat this until the pH of the water
above the phosphocellulose is below 11 (generally this means washing
atleast three times).
4. Wash the phosphocellulose for no more than 5 minutes with 500 mL of 0.5
M HCl, and decant off the excess.
5. Repeat step (3) until the pH of the water above the phosphocellulose is
above 3 (again, this generally means washing atleast three times).
6. Wash with 500 mL 0.5 M phosphate buffer, pH 7.2. Decant off some of the
excess buffer and store the phosphocellulose in this buffer at 4 C until you
are ready to use it. The resin should be used within a week.
1. Pour the phosphocellulose in a glass column and let it equilibrate in buffer B
overnight before the day of the prep. Pour off the potassium phosphate from
the phosphocellulose, and add approximately 250 mL of 1X Buffer B with 50
mM NaCl. Allow the resin to settle, and pour off the excess.
2. Pour the phosphocellulose resin (all that you cycled) into a 500 mL column.
Equilibrate the column with 1X Buffer B with 50 mM NaCl (this will take a
several hours). You can begin preparing the crude extract while the column
is equilibrating.
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PROTEIN PREP:(this procedure takes a week. It is better to start this on
Monday or Tuesday morning. Day 1 is most intense.)
Day 1: Today you will prepare the crude extract, load the crude extract onto the
phosphocellulose column.
1. Early in the morning, remove the cells from the -80 C freezer and place them
on the desktop. They will take awhile to thaw.
2. Add 200 - 300 mL of 1X Buffer A to the 100 mL of previously suspended cells
and stir with a glass rod and stir bar until cells are evenly distributed. DO
THIS ON ICE.
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3. Add 0.4 mg of lysozyme per mL crude extract, and 1 mM fresh PMSF (to
make a 0.1 M solution of PMSF, add 87 mg of PMSF to 5 mL isopropanol.
add the desired volume so that the final concentration is 1 mM. (3 - 4 mL in
this case)). Stir on ice for 30 minutes.
4. Put the cells into polypropylene tubes (50 mL). Lyse the cells with three
cycles of freezing with liquid nitrogen and thawing with a 37 C water bath.
(Mix cells by hand-shaking in the 37C and remove as soon as it is
thawed).
5. Divide the suspension into ultracentrifuge tubes and untracentrifuge at
45,000 rpm (check rotor speed-limit) for 1 hour to pellet the cell debris and
nucleic acids. Make sure the rotor is chilled to 4 C prior to centrifugation.
6. Pour the supernatant into a beaker (do not include any thick slimy stuff) and
keep it at 4 C until you are ready to load it onto the column. Save a small
fraction (100 L) of the supernatant for analysis by SDS-PAGE.
7. Before loading the column, make sure that it is equilibrated. This is done by
checking the pH and the conductance of the eluent from the column and
comparing it to the pH and the conductivity of the buffer going into the column
(1X Buffer B with 50 mM NaCl). The pH and the conductivity of the eluent
should be the same as the input. Do not begin loading the column until
these conditions are reached.
8. Dilute the crude extract with 1X Buffer B no salt until the conductance of this
solution is less than the conductivity of 1X Buffer B with 50 mM salt (input).
9. Make sure that you zero the UV detector while running buffer through the
column. When the column is equilibrated, you can begin loading the column.
Maintain a flow rate of 1 - 1.4 mL/min to avoid compressing the column, and
collect the eluant in a beaker.
10. When the protein is loaded, wash the column with 1X Buffer B with no salt.
Wash the column until the UV absorbance peak returns to baseline. This can
be an overnight step). Collect the wash in a separate beaker from the load
(save both until the end and for analysis by SDS-PAGE).
11. Day 2: Today you will run the gradient on the phosphocellulose column to
elute the protein, pour the DEAE column, equilibrate the DEAE column, and
run an SDS-PAGE gel to analyze the fractions from the phosphocellulose
column.
12. Run a linear gradient from 1X Buffer B with 50 mM NaCl to 1X Buffer B with
500 mM NaCl to elute the protein from the phosphocellulose. The total
volume of the gradient will be 800 mL, so make sure that you have at least
400 mL of each buffer. Collect 10 - 15 mL fractions.
13. While the gradient is running, make 2 gels for SDS-PAGE. They should be
10% running gel, and 5% stacking gel (use the comb with 15 lanes). You will
use one after the phosphocellulose column, and one after the DEAE column.
Wrap the gels in saran wrap and store them at 4 C until you are ready to use
them. Again, while the gradient is running, you can pour the DEAE column
and start equilibrating it. Pour approximately 80 mL of the DEAE into the
column, and equilibrate it with 1X Buffer C with 100 mM NaCl (you can begin
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equilibrating it by gravity while the pump is being used for the gradient).
Equilibration is complete when the conductivity of the eluent is the same or
lower as the input (1X Buffer C with 100 mM NaCl). Store the column in the
cold room overnight until you are ready for it (make sure it does not run dry).
14. A peak should elute from the phosphocellulose column before or about halfway through the gradient. When the gradient is finished, choose 11 fractions
that you wish to run on the SDS-PAGE gel (make sure to take samples at the
end of the peak to determine the exact cut off).
15. Run a 10% SDS-PAGE gel. Make sure to include the crude extract, the
eluent from the load, the eluent from the wash, and a standard protein. This
leaves 11 lanes for fractions. Stain the gel in Coomassie blue and allow it to
completely destain overnight.
16. Day 3: Today you will load the DEAE column, wash the column, and run the
gradient.
17. Look at the SDS-PAGE gel from yesterday and determine which fractions
contain the protein. Pool the fractions where the protein is most abundant,
making sure to cut off as many impurities as possible.
18. Dilute the protein solution with 1X Buffer C no salt until the conductivity is
lower than 1X Buffer C with 100 mM NaCl.
19. Assuming the DEAE column is equilibrated, load the protein on the column.
Collect the eluent in a beaker.
20. Wash the DEAE column for 2 - 4 hours 1X Buffer C with 100 mM NaCl.
Collect the eluent from the wash in a separate beaker from the load.
21. Run a gradient from 1X Buffer C with 100 mM NaCl to 1X Buffer C with 550
mM NaCl. The total volume of the gradient will be 800 mL so make sure that
you have atleast 400 mL of each buffer. Collect 10 - 15 mL fractions. (This
step can be done overnight).
22. Day 4: Today you will run an SDS-PAGE gel and concentrate the protein.
23. Analyze the fractions by SDS-PAGE as described on Day 2, step 5.
24. While the gel is running, soak the concentrator filter (YM30 Amicon) in
distilled water (shiny side down) for atleast an hour with atleast 3 changes of
water.
25. After looking at the SDS-PAGE gel, pool the purest and most abundant
fractions for concentration.
26. Place the filter in the bottom of the concentration chamber (shiny side up)
and seal the bottom of the chamber.
27. Add the pooled DEAE fractions into the concentrator. Save a small fraction
of the pooled fractions and determine its concentration (roughly) by UV
absorbance. Knowing that you want to concentrate the protein to
approximately 100 -200 M, determine the final volume to which you should
concentrate the protein.
28. Assemble the concentrator in the cold room or outside in an ice-bath. Attach
the hose to the pressurized nitrogen tank, and slowly increase the pressure in
the concentrator to 20 psi.
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29. Collect the drops that come out of the concentrator into a graduated cylinder
so that its volume can be measured. Measure the UV absorption of the
solution coming out of the concentrator and confirm that it is close to the
absorbance of Buffer C.
30. Continue concentrating for several hours until the desired volume is reached
(or stop if the solution becomes opaque).
31. When complete, shut off the pressure slowly. You can use a spin column to
further concentrate the protein if needed.
32. Aliquot the concentrated protein into microfuge tubes. Store the microfuge
tubes at -80C.
33. Perform a Bradford Assay and take UV absorption (in 8M urea, the extinction
coefficient at 280 nm is 83,600 M-1cm-1 for gp4A' protein) to calculate the final
concentration of the protein. Label the tubes.
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