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 .
