Purification of Papillomavirus Capsids Using
Agarose Gel Filtration
revised Feb 2014
Laboratory of Cellular Oncology, NCI
Citation: Buck et al (2008) “Arrangement of L2 within the papillomavirus capsid” J. Virol82:5190, PMID: 18367526
Citation: Buck & Thompson (2007) “Production of papillomavirus-based gene transfer vectors” Current Protocols in Cell BiologyUnit 26.1, PMID: 18228512
This protocol describes a method for rapidly purifying papillomavirus capsids out of crude cell lysates or Optiprep. The small-scale (5 ml column) protocol uses gravity flow, but in principle it could be scaled up for use with FPLC. A simplified method using spin columns (see below) offers higher throughput at the cost of somewhat reduced separation efficiency.
Gel filtration (size exclusion chromatography) effectively removes small solutes, including detergents, salt, nucleases, Optiprep and the majority of cellular proteins. Capsid recovery is typically >80%. Although the purity of agarose gel-filtered capsids isn’t quite as good as Optiprep-purified capsids, it is not as physically disruptive as ultracentrifugation.
The protocol was initially developed to purify fragile immature HPV16 capsids out of clarified cell lysates. When we initially attempted to use agarose bead columns equilibrated with plain PBS (150mM NaCl) we observed severe precipitation of the immature capsids. In fact, even fully mature capsids tended to aggregate on plain PBS bead columns. Three different modifications were adopted to combat the denaturation of capsids. First, NaCl was increased to a total of 500mM. An even more important modification was pre-blocking the agarose beads with an overnight incubation in 1% BSA. This incubation presumably blocks disruptive non-specific interactions between the capsid and the resin.
A group at Merck has a nice paperexamining the problem of capsid aggregation and how to combat it. The paper shows that small amounts of certain detergents, such as Tween-80 (Sigma# P6349) can prevent capsid aggregation by blocking disruptive interactions between capsids and solid surfaces. Adding Tween-80 (Sigma# P6349) to the column wash/elution buffer at 0.01% (v/v) can help combat aggregation (i.e., there may be an additive protective effect if Tween-80 is used after the BSA blocking). Tween-80 is not particularly cytotoxic, so can readily be used for purification of pseudovirions that will ultimately go on cells. Using only Tween-80 (instead of BSA) was not as effective for gentle purification of immature capsids. In other words, BSA-blocking the column seems to be the most important factor (and adding Tween-80 can be considered optional).
Materials:
•5ml plastic columns(Pierce #29922) (also available in 2ml and 10ml sizes). 20 ml columns are available from Bio-Rad (Cat# 732-1010)
•2% agarose beads(50-150µm diameter), Agarose Bead Technologiescat# A-1020-S
Alternative: 1.4% agarose beads, http://www.bioscience-beads.com/
Overall purity using 1.4% beads isn’t quite as good but separation between mature and immature/non-infectious capsids might be slightly better. We’ve never tried using 1.4% beads for polyomaviruses. They might not be suitable (polyomavirus capsids are smaller).
•DPBS/0.5M NaCl: supplement Dulbecco’s PBS (e.g., Invitrogen #14040-141) with 1/14thvolume of 5M NaCl. If DPBS is unavailable, mix:
500ml plain PBS
225 ul of 2M CaCl2
125 ul of 2M MgCl2
1050 ul of 1M KCl
35 ml of 5M NaCl
Then sterile filter.
•DPBS/0.5M NaCl supplemented with 1% (w/v) BSA (Fraction V, Sigma)
•Optional: DBPS/0.5M NaCl supplemented with 0.01% Tween-80
Packing the column (perform one day in advance)
1) Put the plastic column in a clamp stand. Place the bottom cap on the column.
2) Completely fill the column with water (or plain PBS). Briefly remove the bottom cap to eject any bubbles trapped in the bottom of the column.
3) Optional: add 100 µl of 10% Triton (or Brij). Detergent can make it easier to remove air from the frit.
4) Float a frit on the surface of the water. Tap frit down to the bottom of the column using the wide end of a 2ml aspirating pipet (or the serum separator provided with the column kit).
4) Remove the bottom cap and drain out nearly all of the water.
5) Suspend the agarose beads by gently swirling the bottle. Pour slurry to almost fill the column. Allow the slurry to settle 5 minutes.
6) Remove the bottom cap and allow the bead storage buffer to drain out. At this point in the protocol it doesn’t really matter if the surface of the bead bed is allowed to briefly dry out.
7) Equilibrate the column by adding one bed volume of water (or plain PBS). This will wash away the ethanol solution the beads are stored in.
8) Add two bed volumes of DPBS/0.5M NaCl with 1% BSA. Note: do not add Tween-80 at this step – detergent might interfere with the BSA-blocking. This would be problematic, since the blocking effects of Tween are almost instantaneously reversible once the Tween is removed, whereas BSA blocking seems very slow to reverse.
9) After equilibrating the beads into DPBS/0.5M NaCl/1% BSA, replace the bottom cap. Seal the top of the column with parafilm and resuspend the beads.
Note: It is important to resuspend the beads after equilibration with room temperature buffer. Chilled solutions contain more dissolved oxygen, which can form bubbles upon warming to room temperature. Trapped bubbles disrupt flow through the gel bed. It may be helpful to degas the solution used to suspend the beads (see protocol that comes with the Pierce columns). The easiest way to suspend the beads is by putting the parafilm-sealed column on a Nutator rocker for ~15 minutes. If a suitable rocker is not available, the beads can be suspended by gently inverting the column by hand, but this is difficult to do without causing foaming. Foaming makes it difficult to avoid getting air bubbles trapped in the bed or on the plastic housing.
10) Remove parafilm. Remove bottom cap. When the meniscus approaches the narrow portion of the column, put the bottom cap back on. Lodge the frit by pressing it down using a 2 ml aspirating pipet (or a pair of forceps). If the frit is angled, gently tap the higher edge. Leave at least several mm of space between the frit and the gel bed. Do not compress the gel bed.
11) Immediately prior to use, wash the column with ~10 column volumes of DPBS/0.5M NaCl (optional: supplement the wash buffer with 0.01% Tween-80). Washing the column with only 4 column volumes may result in contamination of the capsids with residual BSA. After washing the column, tap the frit down a bit (if necessary). Column is now ready to use.
12) Columns can be stored at room temperature after exchanging into plain PBS supplemented with 0.05% azide. Storing columns at 4ºC may result in bubble formation (see note in step 8). Columns can be regenerated for re-use by extensive washing with DPBS/0.5M NaCl, followed by re-blocking with DPBS/0.5M NaCl with 1% BSA.
Performing the filtration
1) Prepare high density Brij (or Triton) cell lysates (>108cells per ml) according to standard pseudovirus protocol. Clarify lysate as instructed in the main protocol
2) Load clarified supernatant onto the washed column (see above). Do not load more than 10% of the bed volume (e.g., <500µl of lysate on a 5ml column)
3) Elute the column with DPBS / 0.5M NaCl (optional: +0.01% Tween-80). Collect 250µl fractions in siliconized tubes. Collect at least 12 fractions.
4) Screen fractions by BCA protein assay (Pierce #23227) in a 96-well plate. Look for an initial peak of capsids followed by a larger peak of cellular proteins. Capsids with encapsidated DNA can be screened using Picogreen dsDNA reagent.
5) The capsid peak should come out at ~1/3rdof bed volume (~1.75ml of eluate ~fraction 7).
6) Removal of Optiprep can be verified by OD 250/280 (UV) ratio. Pure protein solutions have a 250:280 ratio of about 1, Optiprep has a ratio much greater than 1. Note: using absorbance as a readout, we’ve found that Optiprep is difficult or impossible to remove using standard dialysis procedures. Column chromatography is therefore much preferable.
Simplified procedure using spin columns
150729 Mike Tisza
Materials:
-Pierce 89896 centrifuge columns, 2ml
-Suspension of Sepharose 4B (Sigma 4B200)*
- DPBS/0.8M NaCl/0.1% BSA
Procedure:
1) Place spin columns (with bottom caps removed) into 15ml conicals.
2) Thoroughly resuspend beads in their bottle by rotating/shaking
3) Use a 10ml serological pipet to pipet ~2.5 ml bead suspension (Sepharose 4B) into spin column. The goal is to get a 2 ml column bed volume.
4) Add 1 ml of DPBS/BSA solution to the top of each column.
5) Spin at 735 x g for ~30 seconds. Spin time wasn’t carefully optimized – a shorter spin might be feasible. Longer spins might cause so much drying that the gel bed develops cracks. Although the over-dried bed can be rehydrated, the cracks introduce air bubbles that disrupt flow through the resin. If cracking is observed, fully resuspend the beads in DPBS/BSA and spin for less time and/or at lower speed.
6) Discard flowthrough. To exchange the suspension media with DPBS/BSA, add 2 ml DPBS/BSA solution and spin again for 30 seconds at 735 x g. Optional: repeat this buffer exchange step.
7) At this point, there should be no liquid above the bead bed. Apply 0.3 bed volumes (i.e., typically 600 µl) of your sample to the top of the bed. This ratio of sample to bed volume worked well for separating HPV16 VLPs from Benzonase endonuclease, but it remains conceivable that smaller viruses may travel through column more slowly. You can dilute the sample with DPBS/BSA solution, if necessary to achieve correct volume.
8) Spin the sample-loaded column for 30 seconds at 735 x g
9) Virus will be in the eluate. Smaller solutes will remain in the column.
*Note on resins: Initial experiments tested separation of HPV16 VLPs from myoglobin. Sepharose 4B beads outperformed Toyopearl HW-65F and Sepharose 2B. 2% agarose beads were tested but abandoned because the agarose beads squeezed through the bottom frit and ended up in the eluate.
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