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Large Scale Purification of Wild-Type Virions Using Sarkosyl
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
The method described herein is an adaptation of the method described in Citation #1. Most phage clones displaying a foreign peptide will withstand a detergent treatment. The resistance of the phage to detergent is a key step in the purification process. Wild-type phage give approximately 3 X 1012 virons/ml under the conditions outlined below.
Procedure
A. Preparation of Inoculum of Wild-Type Virons
1. Prepare an overnight culture of K91BluK in NZY Media (or LB Media; see Hint #1).
2. Inoculate 5 ml of NZY Media (or LB Media) in a 1 x 3 inch culture tube with 50 μl of the overnight culture of K91BluK from Step #1. Incubate the culture at 37°C with shaking just until it becomes turbid.
3. Inoculate the culture with a single viron plaque with a sterile inoculating loop or a sterile wooden stick.
4. Continue incubation at 37°C with shaking overnight.
5. A 50 μl aliquot of the overnight colony should contain approximately 1.5 X 1011 virons (3 X 109 virons per μl). Use this solution as the inoculum for Step #B1.
B. Crude Purification of Wild-Type Virons
1. Inoculate 25 ml of NZY Media in a 250 ml side-arm flask with 250 μl of an overnight culture of K91BluKan (see Hint #2).
2. Incubate with vigorous shaking at 37°C until the Optical Density at 600 nm reaches approximately 0.2 (OD600 = 0.2)
3. Add 1 liter of pre-warmed (37°C) NZY Media to each of four 3.8-liter fernbach flasks and add 25 ml to a 250 ml side-arm flask.
4. Use 2.5 ml of K91BluKan to inoculate each of the four fernbach flasks and use 15 ml of K91BluKan to inoculate the side-arm flask.
5. Inoculate the side-arm flask with approximately 2 X 1011 wild-type virions in 200 μl or less.
6. Continue to incubate all five flasks (the four fernbach flasks and the 250-ml side-arm flask) with shaking until the OD600 of the side-arm flask reaches 0.6.
7. Pour the infected culture from the 250-ml side-arm flask into a disposable 50-ml conical centrifuge tube and pipette 3 ml into each of the four fernbach flasks. Continue to incubate the fernbach flasks with vigorous shaking overnight at 37°C.
8. Add approximately 350 ml of the infected culture into a 500 ml centrifuge bottle and centrifuge at 5,000 rpm in a Sorvall™ GS-3 rotor (4,200 X g) for 10 min.
9. Pour the supernatant into pre-weighed 500 ml centrifuge bottle (see Hint #3).
10. Centrifuge the infected culture pellet again at 8,000 rpm in a Sorvall™ GS-3 rotor (10,800 X g) for 10 min.
11. Combine the supernatant with the previously collected supernatant and estimate the volume in each tube (see Hint #4).
12. Add 0.15 volume of PEG/NaCl to each bottle and mix well.
13. Incubate at room temperature overnight to pellet the phage.
14. Centrifuge the bottles at 8,000 rpm in a Sorvall™ GS-3 rotor (10,800 X g) for 20 min.
15. Discard the supernatant and centrifuge the pellet again at 8,000 rpm in a Sorvall™ GS-3 rotor (10,800 X g) for 5 min.
16. Aspirate the supernatant.
17. Add 10 ml of TBS Solution to each bottle and incubate at room temperature with constant shaking to resuspend the phage pellet.
18. Pool all phage-containing solutions (approximately 120 ml) into a single 250 ml conical centrifuge bottle. This volume is 1 Volume.
19. Centrifuge at 12,000 rpm in a Sorvall™ GSA rotor (24,000 X g) for 15 min to pellet any insoluble material. Transfer the supernatant into a fresh 250 ml centrifuge bottle.
20. Add 13.3 ml (one-ninth volume) Triton-X 100 Solution to the supernatant in Step #9. Incubate with gentle shaking at room temperature for 1 hr.
21. Add 20 ml PEG/NaCl (one-sixth volume) to the solution from Step #20 and mix well by inverting several times.
22. Centrifuge at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) for 15 min.
23. Discard the supernatant and centrifuge the pellet again at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) for 7 min.
24. Aspirate the supernatant.
25. Dissolve the pellet in 60 ml of TBS Solution by shaking at room temperature.
26. When the phage pellet is completely dissolved, centrifuge at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) for 10 min.
27. Transfer the supernatant into a fresh 250 ml bottle.
28. Add 60 ml of 2% Sarkosyl Solution and gently incubate with gentle shaking at room temperature for 1 hr.
29. Add 18 ml of PEG/NaCl and mix by inverting several times.
30. Centrifuge at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) at room temperature for 15 min.
31. Discard the supernatant and centrifuge the pellet again at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) for 15 min.
32. Aspirate the supernatant.
33. Dissolve the pellet in approximately 100 ml of TBS Solution by shaking at room temperature.
34. Centrifuge the viron solution at 10,000 rpm in a Sorvall™ GSA rotor (17,000 X g) for 10 min and decant the supernatant into a clean centrifuge bottle.
C. Cesium Chloride Purification of Wild-Type Virions
1. Place a clean 250 ml glass beaker onto a scale and tare the weight to zero.
2 Add 29.70 g of Cesium Chloride (CsCl) to the beaker and tare the weight to zero again.
3. Add 66.11 g of viron solution to the beaker.
4. Pour or pipette the cleared supernatant (prepared in Step #B34) into the beaker. If necessary, add additional TBS Solution to bring the net weight of the aqueous solution to 66.11 g.
5. Stir gently to dissolve the CsCl (see Hint #5).
6. Divide the solution into two equal parts and add each solution into a 1 x 3.5 inch centrifuge tube appropriate for an SW28 rotor.
7. Centrifuge the solution at 25,000 rpm in a Beckman™ SW28 rotor (83,000 X g) at 15°C for 36 to 48 hr (see Hint #6).
8. Remove the solution above the phage bands with a sterile transfer pipette while taking care to keep the tip of the pipette at the meniscus.
9. Remove the phage bands with a fresh sterile transfer pipette and pool them into a single 50 ml sterile disposable conical centrifuge tube.
10. Bring the total volume in each 50 ml tube to 40 ml with TBS Solution; then pipette 5 ml into each of eight Beckman™ SW 60Ti bottles.
11. Fill the bottles to the shoulder with TBS Solution and centrifuge at 50,000 rpm with a Beckman™ SW 60Ti rotor (257,000 X g) for 4 hr at 4°C.
12. Discard the supernatant and centrifuge the pellet again at 50,000 rpm with a Beckman™ SW 60Ti rotor (257,000 X g) for approximately 20 min.
13. Aspirate the supernatant.
14. Add 10 ml of TBS Solution to each bottle, wrap the tops securely with parafilm, and incubate with rotation overnight at 4°C to dissolve the phage pellet.
15. Fill each bottle to the shoulder with TBS Solution and mix well.
16. Centrifuge at 50,000 rpm with a Beckman™ SW 60Ti rotor (257,000 X g) for 4 hr at 4°C.
17. Discard the supernatant and centrifuge the pellet again at 50,000 rpm with a Beckman™ SW 60Ti rotor (257,000 X g) for approximately 20 min.
18. Aspirate the supernatant.
19. Add 6 ml of TBS Solution to each bottle, vortex, wrap the tops securely with parafilm, and incubate with rotation overnight at 4°C to dissolve the phage pellet.
20. Vortex the solution and centrifuge briefly in a clinical centrifuge at medium speed to drive the solution off the inner tube walls.
21. Pool all eight solutions into a single 50 ml sterile, conical centrifuge tube.
22. Load the viron solution into dialysis tubing of an appropriate length (Spectrapore, 12 to 14 KDa molecular weight cut off) and dialyze overnight at 4°C against ddH2O. Exchange the ddH2O several times.
23. Transfer the dialysate into a tared 60 ml Nalge bottle and record the weight in grams (which will equal the volume in ml).
24.Combine 5 μl of the phage solution with 495 μl of ddH2O in a sterile 1.5 ml microcentrifuge tube (Step #B13), and mix well (a 1:100 dilution).
25. Remove 400 μl of the 1:100 dilution and scan the absorbance from 240 nm to 320 nm by using ddH2O as a blank (see Protocol ID#2174).
26. Calculate the actual undiluted physical particle concentration by multiplying the net A269 (of the 1:100 dilution) by 9.3633 X 1014 (see Hint #7).
27. Dilute the remaining (100 μl) of 1:100 dilution to 1 X 10-9 (the overall dilution of the viron is 1 X 10-11) with TBS/Gelatin Solution.
D. Plating for Plaque Efficiency
1. Inoculate approximately 5 ml of NZY Media in a 1 x 3 inch culture tube with an overnight culture of K91BluKan.
2. Incubate at 37°C until the culture reaches mid-log phase (when the culture is turbid).
3. Re-liquify TB Soft Agar in a microwave or boiling water bath and dispense approximately 3 ml into two sterile glass 13 x 100 mm tubes.
4. Place the tubes in a 50°C heating block to keep the Agar molten.
5. Pipette 75 μl of the 1 X 10-11 dilution into one sterile 15 ml tube and 75 μl of the TBS/Gelatin Solution into another (as a control).
6. Add 400 μl of the K91BluKan culture to each tube.
7. Combine the 3 ml of molten Soft Agar and 475 μl of the viron/K91BluKan culture onto a fresh culture plate.
8. Combine the 3 ml of molten Soft Agar and 475 μl of the TBS Gelatin/K91BluKan culture (control solution) on a fresh culture plate.
9. Allow the Soft Agar to harden and incubate the plates in a 37°C incubator overnight.
10. Record the number of plaques and calculate the undiluted titer by multiplying the number of blue plaques on the non-control plate by 1.333 X 1012 (see Hint #8).
Solutions
TB Soft Agar
0.5 g NaCl
1 g Bacto Tryptone
Store at room temperature
Add 100 ml ddH2O
Autoclave
Into a number of glass 125-ml bottles weigh:
0.75 g Bacto Agar 
TBS/Gelatin Solution
Store at room temperature
0.1 g Gelatin
Autoclave
100 ml 1X TBS Solution
After autoclaving, swirl to mix in the melted gelatin
2% Sarkosyl Solution
Prepare in 1X TBS Solution
2% (w/v) Sarkosyl
TBS (10X)
Store at room temperature
500 mM Tris-HCl pH 7.5
1.5 M NaCl
Triton-X 100 Solution
1:9 (v/v)Triton-X 100:ddH2O
PEG/NaCl
116.9 g NaCl
Store at 4°C
Stir until the solutes dissolve (it may be necessary to heat to 65°C briefly to dissolve the last crystals of PEG)
475 ml ddH2O
100 g PEG 8000 (Union Carbide)
LB Media (1X)
5 g/liter Yeast Extract
10 g/liter NaCl
Adjust pH to 7.0 with NaOH
Autoclave, store at room temperature
10 g/liter Bacto-Tryptone
5 M NaOH
NZY Media (10X)
Adjust the pH to 7.5 with 5 M NaOH
50 g NaCl
Store at room temperature
100 g NZ Amine A (Humko Sheffield Chemical)
Dissolve in 10 liters of ddH2O
Autoclave
50 g Yeast Extract
BioReagents and Chemicals
Gelatin
NZ Amine A
Kanamycin
Sarkosyl
K91BluK
Bacto Yeast Extract
Triton-X 100
Bacto Tryptone
Tris-HCl
Bacto Agar
Sodium Chloride
PEG 8000
Sodium Hydroxide
Protocol Hints
1. The addition of 100 μg/ml Kanamycin to the growth media is optional.
2. The following is a typical time-course for the inoculation of K91BluKan (Section B, Steps #1 to #7): 0 Hr (elapsed time), Inoculate the 25 ml culture with cells (Step #B2); 2 hr 40 min, culture reaches OD600 0.2; 2 hr 55 min; inoculate 1 liter cultures with 25 ml culture and inoculate the 25 ml culture with viron (Steps #B3 to #B5); 3 hr 50 min, culture approaches OD600 of 0.5; 4 hr 25 min, culture reaches OD600 of 0.6, inoculate the 1 liter flasks with viron (Steps #B6 to #B7).
3. Record the weight of each conical centrifuge bottle before adding any supernatant (make sure to mark each centrifuge bottle). Weigh the centrifuge bottle after adding the last of the supernatant. Subtract the full weight from the empty weight to approximate the volume in each centrifuge bottle. Assume that the density of the solution is 1 g/ml.
4. The 4 liters of culture prepared in Steps #B1 to #B7 should now be reduced to twelve 500 ml conical centrifuge bottles.
5. When the salt is completely dissolved, the solution should have a volume of approximately 73.7 ml. The density of the solution should be 1.3 g/ml or a 31% (w/w) solution (31% w/w is based on the following calculation: 29.70 / [29.70 + 66.11]). Confirm that the density of the solution is between 1.29 to 1.31 g/ml.
6. There should be a crisp, clean phage band toward the middle of each tube. The phage band should be free from the flocculent bands that are usually present when phage are not treated with detergent.
7. The contributor of this protocol routinely measures a net A269 of 0.300 corresponding to 2.81 X 1014 virions/ml (or 6.62 mg/ml mature pVIII subunits).
8. Assuming a physical particle concentration of approximately 2 X 1014 virions/ml and 100% infectivity, approximately 150 plaques should appear.
Citation and/or Web Resources
1. Wickner W. Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli. (1975) Proc Natl Acad Sci U S A. 72:4749-53.
