PBCV purification w/proteinaseK
PBCV purification w/proteinaseK

Chlorella cells (1.5-2 107 cells/ml) were inoculated with filter sterilized (0.45 m filter pore size) virus at a multiplicity of infection (MOI) of ~0.001 and incubated for 2-3 days at 25C with continuous light and shaking.

Cell lysates were centrifuged at 4,000 g for 5 min at 4C; Triton (to 1%) was added to the supernatant and kept at room temperature with constant mixing for 1 h.

The sample was then centrifuged at 4C for 50 min at 53,000 g to pellet virus.

The virus pellet was resuspended in a small volume of Tris Buffer (TB: 50 mM Tris-HCl, pH 7.8) and incubated with proteinase K (0.02 mg/ml) for 1 h at 45 C.

The virus suspension was then layered onto 10-40% linear sucrose density gradients equilibrated with TB and centrifuged in a swinging bucket rotor at 4C for 20 min at 72,000 g.

The virus band was removed from the gradient with a sterile needle, diluted with TB and centrifuged for 3 h at 80,000 g.

The virus in the pellet was resuspended and subjected to a second sucrose density gradient centrifugation and sedimentation process to ensure virus purity.

Virus concentrations were determined on a UV spectrophotometer as A260, and by plaque assay (PFU/ml).

One A260 unit of PBCV-1 routinely yields 1.5-2.5 1010 PFU/ml. Isolated and purified virus was stored at 4C.