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Large-Scale Purification of fd-tet-Derived Virions Using Sarkosyl
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
The protocol 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. For cases where the clone (or clones) of interest may be sensitive to detergent (e.g., when purifying an entire library with many different clones), modifications to the protocol are highlighted in bold text.
Procedure
A. Preparation of Inoculum of fd-tet-Derived Virons
1. Inoculate 1 liter of NZY + TET in a Fernbach flask with a well-separated colony and incubate with vigorous shaking overnight at 37°C.
2. Centrifuge the culture in three 500 ml centrifuge bottles at 5,000 rpm for 10 min in a Sorvall™ GS-3 rotor (4,200 X g) and collect the supernatant in a fresh 500 ml centrifuge bottle.
3. Centrifuge the collected supernatant at 8,000 rpm for 10 min in a Sorvall™ GS-3 rotor (10,800 X g), collect the supernatant in a tarred 500 ml centrifuge bottle, and note the net weight (see Hint #1).
B. Crude Purification of fd-tet-Derived Virions
1. Add 0.15 volume of PEG/NaCl to each bottle and mix thoroughly with approximately 100 inversions. Incubate the solution overnight at 4°C to precipitate the phage (see Hint #2).
2. Centrifuge at 8,000 rpm for 20 min in a Sorvall™ GS-3 rotor (10,800 X g) and discard the supernatant.
3. Centrifuge again at 8,000 rpm for 20 min in a Sorvall™ GS-3 rotor (10,800 X g), then carefully remove and discard any remaining supernatant with a pipette.
4. Add 10 ml of TBS to each bottle and incubate at room temperature with shaking in order to resuspend the phage pellet.
5. Pool all the phage (30 ml altogether) in an Oak Ridge centrifuge tube and centrifuge at 10,000 rpm for 15 min in a Sorvall™ SS-34 rotor (16,900 X g) to pellet any insoluble matter.
6. Pour and pipette the supernatant to a fresh Oak Ridge tube. For purification without detergent treatment: Add an additional 3.3 ml of ddH2O and proceed with Step #8.
7. Add 3.3 ml (one-ninth the volume) of Triton X-100 Solution to the Oak Ridge tubes and rock horizontally at room temperature for 1 hr.
8. Add 5 ml of PEG/NaCl (one-sixth the volume) and mix well with many inversions. Centrifuge immediately at 10,000 rpm for 15 min in a Sorvall™ SS-34 rotor (16,900 X g) and discard the supernatant.
9. Centrifuge again at 10,000 rpm for 15 min in a Sorvall™ SS-34 rotor (16,900 X g). Carefully remove and discard any remaining supernatant with a pipette.
10. Dissolve the phage pellet in 15 ml of 1X TBS by pipetting the solution up and down with a 5 ml pipette.
For purification without detergent treatment: Dissolve the phage pellet in 30 ml of 1X TBS rather than 15 ml.
11. Centrifuge the solution at 10,000 rpm for 10 min in a Sorvall™ SS-34 rotor (16,900 X g) and collect the supernatant into a fresh Oak Ridge tube.
For purification without detergent treatment: Proceed directly to Step #12.
12.Add 15 ml of 2% Sarkosyl Solution to the collected supernatant, mix gently by inversion, and then rock horizontally at room temperature for 1 hr.
13. Add 4.5 ml of PEG/NaCl and mix well by many inversions. Centrifuge immediately at 10,000 rpm for 10 min in a Sorvall™ SS-34 rotor (16,900 X g) and discard the supernatant.
14. Centrifuge again at 10,000 rpm for 15 min in a Sorvall™ SS-34 rotor (16,900 X g). Carefully remove and discard any remaining supernatant with a pipette.
15. Dissolve the phage pellet in 10 ml of 1X TBS by pipetting the solution up and down with a 5 ml pipette (see Hint #3).
16. Incubate the solution with shaking at room temperature for 1 hr.
17. Centrifuge the resuspended phage at 10,000 rpm for 10 min in a Sorvall™ SS-34 rotor (16,900 X g) to clear an insoluble material.
18. Collect the supernatant into a 15 ml conical centrifuge tube (see Hint #4).
C. Cesium Chloride Purification of fd-tet-Derived Virions
1. Place a clean 50 ml glass beaker onto a scale and tare the weight to zero.
2.Add 4.83 g of Cesium Chloride (CsCl) to the beaker and tare the weight to zero once again.
3. Add the cleared supernatant plus additional 1X TBS to bring the net weight of the aqueous solution in the beaker to 10.75 g.
4. Stir gently to dissolve the CsCl (see Hint #5).
5. Load the solution into a polyallomer SW41 tubes and centrifuge in a Beckman™ Type SW 40 Ti rotor (173,000 X g) for 36 to 48 hr (see Hint #6).
6. Using a sterile transfer pipette, remove the solution above the phase band. Keep the tip at the meniscus as much as possible. Next, use a fresh transfer pipette to transfer the phage band into a clean 60 Ti centrifuge bottle.
7. Fill the 60 Ti centrifuge bottle to the shoulder with 1X TBS and centrifuge at 50,000 rpm in a Beckman™ Type 60 Ti rotor (257,000 X g) for 4 hr.
8. Discard the supernatant and centrifuge again at 50,000 rpm in a Beckman™ Type 60 Ti rotor (257,000 X g) for approximately 30 min.
9. Carefully remove and discard any remaining supernatant with a pipette.
10. Add 3 ml of 1X TBS, vortex, wrap the tops securely with parafilm, and rotate overnight at 4°C to dissolve the phage pellet.
11. Vortex the solution and centrifuge briefly in a clinical centrifuge at medium speed to drive the solution off the inner tube walls.
12. Load the viron solution into an appropriate length of dialysis tubing (Pierce Slide-A-Lyzer 12 to 14 KDa molecular weight cut off) and dialyze 12 to 24 hr at 4°C against 1 liter of 1X TBS. Exchange the solution three times.
13. Transfer the dialyzed phage into a fresh polyallomer 10 ml Oak Ridge tube and centrifuge at 10,000 rpm in a Sorvall™ SS-34 rotor (16,900 X g) for 5 min.
14. Transfer this solution to a tared 4 ml tube, then store and record the weight in grams (which will equal the volume in ml).
15. Combine 6 μl of the supernatant and 294 μl of 1X TBS (1:50 dilution) in a fresh 500 μl microcentrifuge tube.
16. Scan the 300 μl solution from an absorbance of 240 nm to 320 nm using 1X TBS as the blank (see Protocol ID#2174).
Solutions
5 M NaOH
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)
NZY + TET
20 μg/ml Tetracyline
1X NZY Media
Include 1 mM IPTG if the vector is f88-4
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
Tetracyline
Yeast Extract
NZ Amine A
Triton X-100
Sodium Hydroxide
IPTG
Sodium Chloride
Tris-HCl
PEG 8000
Sarkosyl
Protocol Hints
1. Weigh the 500 ml centrifuge bottle before adding the solution to it. Weigh the bottle again after adding all of the solution to the bottle. Subtract the bottle weight with solution from the bottle weight without solution. The difference in weight is the approximate volume of solution collected (approximately 300 g = 300 ml).
2. To calculate 0.15 volumes, take the approximate volume (calculated in Step #3 and Hint #1) and multiply it by 0.15. This value is the volume of PEG/NaCl to add.
3. Phage from up to two 1-liter cultures can be pooled in the 10 ml of 1X TBS and centrifuged. Additionally, any suitable buffer can be substituted for 1X TBS at this point. For example, when phages are to be used for immunizing animals, a PBS Solution is substituted for TBS in this and all subsequent steps.
4. Partially purified phage can be stored at 4°C.
5. When the salt is completely dissolved, the solution should have a volume of 12 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 clean phage band near the middle of each tube that is quite translucent and difficult to see at first. Its upper boundary is very sharp and is noticeable as a discontinuity in the refractive index even if light scattering from the phage band is very weak. For purification without detergent treatment: If detergent treatment is omitted, there will usually be a sharp, thin flocculent band above the phage band, and another much larger flocculent band just below the phage band. These flocculent bands are relatively opaque and are therefore much more apparent at first glance than the very translucent phage band. If you are unable to see the phage band, it is recommended to take approximately 1 cm just above the lower, larger flocculent band. Avoid the lower flocculent band if possible, although there is little harm if some of this band contaminates the phage band.
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.
