CviRI Purification From XZ-6E Virus Infected NC64A Chlorella
Prepare Buffer A: 
Prepare Buffer B: 
Prepare Buffer C: 
Prepare Storage Buffer: 
Prepare 1X CviRI Assay Buffer: 
Thaw the 3-4 hour XZ-6E virus-infected NC64A chlorella and suspend in MSK flasks with Buffer A.
Suspend with 20 mL per flask per 1.0-1.5 X 1011 infected cells.
Homogenize the cells in the MSK mechanical homogenizer with 15 gm of 0.3 mm glass beads at 4,000 rpm for 90 sec (2 X 45 sec) with CO2 cooling.
Recover the homogenate to clean tubes.
Adjust the homogenate supernatant to 70% saturation with (NH4)2SO4 at 4°C with gentle stirring.
Add the (NH4)2SO4 gradually.
Centrifuge the material in the Sorvall SS34 rotor at 10,000 rpm, 10 min, 4°C.
Suspend the pellets with Buffer A.
Centrifuge the material in the Sorvall SS34 rotor at 10,000 rpm, 10 min, 4°C.
Dilute the supernatant with 3-5 volumes of Buffer B to reduce the NaCl concentration.
Elute the Heparin-Sepharose column with Buffer B using a 0.2-1.2 M NaCl gradient.
Elute the Blue-Sepharose column (or Affi-Gel-Blue column) with Buffer B using a 0.2-2.0 M KOAc gradient.
Dilute the pooled fractions with 2 volumes of Buffer C to reduce the salt concentration.
Elute the Hydroxylapatite column with Buffer C using a 0-1.0 M KHPO4 gradient.
Dilute the pooled fractions with 3-4 volumes of Buffer B.
Concentrate the pooled enzyme by dialysis overnight into storage buffer at 4°C.
Wash the glass beads 3X with 5 mL of Buffer A and combine with the homogenate.
Centrifuge the homogenate in the Sorvall SS34 rotor at 10,000 rpm,20 min, 4°C.
Save the supernatant.
Incubate at 4°C for 60-90 min without stirring.
Save the pellet.
Per mL of suspension add: 0.45 mL of 4 M NaCl and 0.45 mL of 28% PEG 8000 (heated to 65°C).
Mix gently by inversion for 5-10 min.
Save the supernatant.
Load the material overnight onto a Heparin-Sepharose column equilibrated with Buffer B in the cold room.
Assay the column fractions and pool the active fractions.
Dilute the pooled fractions with 3 volumes of Buffer B to reduce the salt concentration.
Load the material overnight onto a Blue-Sepharose column (or an Affi-Gel-Blue column) equilibrated with Buffer B in the cold room.
Assay the column fractions and pool the active fractions.
Load the material overnight onto an Hydroxylapatite column equilibrated with Buffer C in the cold room.
Assay the column fractions and pool the active fractions.
Load the material overnight onto a small Heparin-Sepharose column equilibrated with Buffer B. 
Elute the column with Buffer B containing 2.0 M NaCl.
Collect small fractions (approximately 35 drops per fraction).
Assay the column fractions (only about the first 10-15 fractions) and pool the active fractions.
Store the enzyme at -20°C.
