Restriction Endonuclease Purification From Virus Infected Chlorella
Thaw the virus infected 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.
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.
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.
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.
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.
Load the material overnight onto a Heparin-Sepharose column equilibrated with Buffer B in the cold room.
Elute the Heparin-Sepharose column with Buffer B using a 0-2.0 M KOAc gradient.
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.
Elute the Blue-Sepharose column with Buffer B using a 0-2.0 M KOAc gradient.
Assay the column fractions and pool the active fractions.
Dilute the pooled fractions with 10-15 volumes of Buffer B, pH 8.5 to reduce the salt concentration.
Load the material overnight onto a Q-Sepharose column equilibrated with Buffer B, pH 8.5 in the cold room.
Elute the Q-Sepharose column with Buffer B, pH 8.5 using a 0-2.0 M KOAc gradient.
Assay the column fractions and pool the active fractions.
Concentrate the pooled enzyme by dialysis overnight into storage buffer at 4°C.
Add BSA (10 mg/mL) to a final concentration of 100 µg/mL.
Save the supernatant.
Incubate at 4°C for 60-90 min without stirring.
Save the pellet.
Save the supernatant.
Store the enzyme at -20°C.
