CviJI Purification From IL - 3A Virus Infected NC64A Chlorella
Prepare Buffer A :
Prepare Buffer B :
Prepare Buffer B , pH 8.5 :
Prepare Storage Buffer :
Prepare 1X CviJI Assay Buffer :
Store the enzyme at - 20°C .
Thaw 7 hour IL - 3A virus infected NC64A chlorella and suspend in MSK flasks with Buffer A .
Recover the homogenate to clean tubes .
Adjust the homogenate supernatant to 70 % saturation with ( NH4 ) 2SO4 at 4°C with gentle stirring .
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 10 - 15 volumes of Buffer B to reduce the NaCl concentration .
Elute the Heparin - Sepharose column with Buffer B using a 0 - 2.0 M KOAc gradient .
Elute the Blue - Sepharose column with Buffer B using a 0 - 2.0 M KOAc gradient .
Dilute the pooled fractions with 10 - 15 volumes of Buffer B , pH 8.5 to reduce the salt concentration .
Elute the Q - Sepharose column with Buffer B , pH 8.5 using a 0 - 2.0 M KOAc gradient .
Concentrate the pooled enzyme by dialysis overnight into storage buffer at 4°C .
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 .
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 10 - 15 volumes of Buffer B to reduce the salt concentration .
Load the material overnight onto a Blue - Sepharose column equilibrated with Buffer B in the cold room .
Assay the column fractions and pool the active fractions .
Load the material overnight onto a Q - Sepharose column equilibrated with Buffer B , pH 8.5 in the cold room .
Assay the column fractions and pool the active fractions .
Add BSA ( 10 mg / mL ) to a final concentration of 100 µg / mL .
