Jun 15, 2018
Purification of Cafeteria roenbergensis virus particles
- 1Max Planck Institute for Medical Research, Heidelberg, Germany
- VERVE Net
- Protist Research to Optimize Tools in Genetics (PROT-G)
Protocol Citation: Matthias Fischer 2018. Purification of Cafeteria roenbergensis virus particles. protocols.io https://dx.doi.org/10.17504/protocols.io.qz2dx8e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 15, 2018
Last Modified: June 15, 2018
Protocol Integer ID: 13082
Keywords: giant virus, CroV, virus purification, Optiprep
Abstract
The concentration and purification of giant DNA viruses from bacteria-containing cultures can present problems because the particle size of giant viruses overlaps with that of smaller bacteria, hence filtration as the only separation method is not possible.
In this protocol, a combination of centrifugation, tangential flow filtration, and density-gradient ultracentrifugation is used to concentrate and purify particles of the giant marine virus CroV that infects the heterotrophic flagellate Cafeteria roenbergensis.
Materials
MATERIALS
Optiprep ( Iodixanol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D1556-250ML
F/2 mediumCatalog #MKK50L
Sorvall Lynx 4000 CentrifugeThermo Fisher ScientificCatalog #75006581
BD Bacto™ Yeast Extract Becton Dickinson (BD)Catalog #212750
Cafeteria roenbergensis culture, from culture collections such as Roscoff or CCAP
SW40 Ti (with Beckmanultracentrifuge) rotor with ultra-clear tubes (14x95 mm)Beckman CoulterCatalog #344060
Slide-A-Lyzer 3 mL Dialysis Cassettes, 20 kDa MWCOThermo Fisher ScientificCatalog #66003
Vivaflow 200 tangential flow filtration unit (0.2 µm, PES), including size 16 tubing for Masterflex peristaltic pumpSartoriusCatalog #VF20P7
Grow cultures of Cafeteria roenbergensis to an initial density of 7E+05 to 1E+06 cells/mL in f/2 artificial seawater medium containing 0.03% (w/v) yeast extract to stimulate bacterial growth (Cafeteria feeds on the bacteria).
If needed, dilute denser cultures to the desired cell concentration with f/2-yeast medium.
Aliquot the cultures, e.g. 50 ml aliquots per 250 ml polycarbonate Erlenmeyer flask, or 500 ml per 3 L polycarbonate Fernbach flask.
Infect host cultures with a Cafeteria roenbergensis virus (CroV) suspension at a multiplicity of infection (moi) of ~0.01.
Incubate at 20-23 °C (constant shaking not required).
Leave one culture uninfected as a negative control.
Monitor host cell density daily by counting on a hemocytometer until lysis occurs (e.g. at 4-6 days post infection).
When cell densities of infected cultures drop below 1E+05 cells/ml, centrifuge the lysates to remove cell debris.
Use 1 L centrifuge bottles, e.g. in a Sorvall F9-6x1000 rotor (used with Sorvall Lynx centrifuges), at a speed of 8000 rcf for 20 minutes and a temperature of 4 °C.
Save the virus-containing supernatant and discard the pellets.
Concentrate the CroV-containing supernatant by tangential flow filtration, e.g. in a Vivaflow 0.2 µm PES unit.
Concentration of up to 15 L is possible with a single Vivaflow unit (can take several days).
Cool the unit and reservoir during concentration, check the tubing regularly for wear and make sure the connections are tight.
Do not exceed a back pressure of 2 bars.
The filtrate is discarded, the CroV-bacteria mixture is concentrated to a final volume of 30-50 ml.
Color may vary depending on the bacteria present in the host cultures.
Save the concentrate in a 50 mL Falcon tube.
To remove most of the bacteria and further concentrate CroV particles, use ultracentrifugation on a two-step Optiprep cushion.
Fill Beckman SW40 ultraclear tubes with ~6 ml of CroV-bacteria concentrate, then slowly underlay with 2.5 mL of a 23% Optiprep solution in 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2, 400 mM NaCl, and then with 1 mL of a 40% Optiprep solution in 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2, 400 mM NaCl. The 23% cushion will stop the migration of bacteria, the 40% cushion will stop CroV particles and pellet them gently.
Fill the tubes carefully to the top with CroV-bacteria concentrate and balance them against each other.
Centrifuge in a Beckman SW40 swinging bucket rotor (always load all buckets, even if empty!) at 100,000 rcf for 2 hours at 15 °C.
Collect the opaque CroV-containing band at the 23%-40% interface by puncturing the tube with a 20G needle and syringe (1-2 mL per tube).
Dialyze the virus particles in 3 mL dialysis cassettes (Slide-A-Lyzer, 20 kDa cutoff) against 1 L of 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 400 mM NaCl at 4°C overnight.
Volume in the dialysis cassette will increase 2-3fold.
Prepare 10%-50% continuous Optiprep, 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 400 mM NaCl gradients in SW40 ultraclear tubes, e.g. using the Gradient Master (settings:81.5° angle, 35 rpm, 1 min 15 sec). Mark tubes at upper half edge and fill with 6.5 mL 10% Optiprep in 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 400 mM NaCl.
Load up to 2 mL of dialyzed CroV suspension per tube by removing gradient solution from the top and replacing it with virus suspension.
Centrifuge for 4 h in a SW40 rotor at 100,000 rcf, 4°C.
Extract CroV-containing bands using a 21G needle and syringe.
Dilute the CroV-Optiprep suspension 3-fold with 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 400 mM NaCl and spin in a tabletop centrifuge for 20 min, 4°C, 10,000 rcf. Lower g-forces and longer centrifugation times are required if the particles should all be intact, e.g. for structural studies.
Save supernatant, dissolve opaque pellets in 50 µl of 50 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 400 mM NaCl, combine the dissolved pellets and spin them again for 15 min at 8,000 g, 4°C.
This should result in a clearly visible white pellet. A yellow color indicates the presence of free lipids, e.g. released from broken virus capsids.
Carefully resuspend the virus pellet..
The saved supernatants can be centrifuged again for 1 h at maximum speed (20,000 rcf), 4°C to collect the remaining virus particles, e.g. for DNA extraction.