VirionPurification.DOC Page 1 10/06/2018
Large-Scale Purification of fd-tet-Derived Virions With or WithoutDetergent Treatment
NOTE: This protocol is adapted from Wickner, W., Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli, Proc. Natl. Acad. Sci. USA72, 4749–4753, 1975. It yields highly purified phage, presumably by desorbing traces of adherent proteins. Most phage clones displaying foreign peptides withstand the detergent treatment in the procedure without loss of infectivity. However, that finding by no means precludes the possibility of subtle alteration of virion structure. For cases where detergent treatment is inadvisable—for example, when purifying a whole library with many different clones—we also show in boxes the modifications required if you want to omit detergent treatment.
1. Use a well-separated colony to inoculate 1 liter of NZY with 20 µg/ml tetracyline (plus 1 mM IPTG if the vector is f88-4; see vectors.doc) in a Fernbach flask; shake vigorously overnight at 37°.
2. Clear by two centrifugations in three 500-ml centrifuge bottles at 5 and 8 Krpm for 10 min in the GS3 rotor (or equivalent). Pour the final supernatants into tared 500-ml centrifuge bottles and note the net weight in each (should be ~300 g = 300ml).
3. To each bottle add 0.15 volume PEG/NaCl. Mix thoroughly by ~100 inversions. Allow to precipitate overnight in cold.
4. Centrifuge bottles 8 Krpm 20 min in the GS3 rotor; RRR; to each bottle add 10 ml TBS; shake bottles in the cold(room temperature OK) to resuspend phage. Pool all the phage (30 ml altogether) in a 50-ml tube. Centrifuge at 10 Krpm for 15 min in the FiberLite rotor[1] to pellet insoluble matter. Pour and pipette the supernatant to a fresh 50-ml tube.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: Add an additional 3.3 ml water to the supernatant and skip directly to step 6.
5. To the 50-ml tube add 3.3 ml (1/9 vol) of a 1:9 v/v mixture of Triton-X100:water. Rock the tube on its side at room temperature for 1 hr.
6. Add 5 ml PEG/NaCl (1/6 vol). Mix by many inversions. Centrifuge immediately at 10 Krpm for 15 min. RRR. Dissolve phage in 15 ml TBS by pumping and stirring with a 5-ml pipette tip.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: Dissolve in 30 ml TBS rather than 15 ml.
7. Centrifuge at 10 Krpm 10 minutes, and pour the supernatant into a fresh 50-ml tube.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: Skip directly to step 9.
8. Add 15 ml TBS containing 2% sarkosyl; mix by gentle inversion; rock the tube on its side at room temperature for 1 hr.
9. Add 4.5 ml PEG/NaCl. Mix by many inversions. Centrifuge immediately at 10 Krpm 10 min. RRR. Dissolve the phage in 10 ml TBS by pumping and scraping with 5-ml pipette tip then shaking in shaker incubator for 1 hr at room temperature. NOTE: Phage from up to two 1-liter cultures can be pooled in the 10 ml and centrifuged in a single SW41 tube below. Any suitable buffer can be substituted for TBS at this point; for example, when phage are to be used for immunizing animals, D-PBS is substituted for TBS in this and all subsequent steps.
10. Centrifuge at 10 Krpm for 10 min in the FiberLite rotor to clear insoluble matter. Pour the supernatant into a 15-ml conical centrifuge tube. Phage can be stored in refrigerator at this point.
11. Into a tared 50-ml beaker weigh 4.83 g CsCl.
12. Tare the beaker again. Add the cleared supernatant plus sufficient additional TBS to bring the net weight of aqueous phase to 10.75 g. Stir gently to dissolve the salt. You should now have 12 ml of a 31% w/w (1.30 g/ml) solution. Confirm that the density is 1.29–1.31 g/ml.
13. Load phage suspension into a clear SW41 tube[2]; centrifuge in SW41 rotor for 36–48 hr at 37,000 rpm. There should be a clean phage band toward middle of each tube; this band is quite translucent and can be difficult to see at first. Its upper boundary is very sharp, and is distinctly noticeable as a refractive index discontinuity even if light scattering from the phage band is very feeble.
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 can’t see the phage band, it’s usually safe to take ~1 cm just above the lower, larger flocculent band. We try to avoid the lower flocculent band if possible, but there’s little harm if some of this band contaminates the phage band.
14. Using a sterile transfer pipette or an electronic pipetter, remove the solution above the phage band, trying as much as possible to keep the tip at the meniscus; then use a fresh transfer pipette or pipette tip to remove the phage band into a 4-ml vial or 50-ml tube.
15. Dialyze in a 3-ml Pierce Slide-A-Lyzer (or other form of dialysis membrane; any molecular weight cutoff should be OK) against three changes of ~1 liter of cold TBS.
16. Remove supernatant to a clean 4-ml vial or other suitable vessel; store in the refrigerator.
17. In a 500-µl Ep tube make 300 µl of a 1/50 dilution. Scan from 220–320 nm. Calculate the undiluted physical particle concentration (see AbsorptionSpectrum.doc).
18. It’s usual to check the infectivity of the virions (TU/virion) by titering tetracycline transducing units as in TUtiter.doc. The infectivity will vary from one titering to another, but will typically be ~5% (10 times lower than wild-type virions).
[1] These excellent rotors allow you to spin standard disposable 50-ml conical screw-cap centrifuge tubes at 15,000 rpm! The pellet collects at the angle rather than the tip, which makes it particularly easy to free of residual supernatant. If you don’t have this rotor, you can use OakRidge tubes and the Sorvall SS34 rotor (or equivalent Beckman rotor) instead.
[2] The capacities of different SW41 ultracentrifuge tubes differ slightly. You may have to adjust the volume upward or downward as necessary. This can be accomplished by changing the total volume and the weight of CsCl in proportion; alternatively, if 12 ml is insufficient to fill the tube to within ~2.5 mm of the rim, a small volume of a separate 31% w/w CsCl solution can be added (no need to mix with the bulk phage/CsCl solution).