Preparation of PBCV-1 Lysin
Centrifuge the NC64A chlorella (use actively growing cells approximately 1.0-2.0 X 107 cells/mL) in the Sorvall GSA rotor at 7,000 rpm, 5 min, 4°C.
Use sterile bottles to harvest the cells.
Discard the supernatants.
Resuspend the cells with sterile BBM.
Adjust the final volume to 500 mL with sterile BBM.
Add filter-sterilized tetracycline (12.5 mg/Ml stock, add 0.1 mL per 50 mL of BBM solution) and PBCV-1 at an moi (multiplicity of infection) of 0.01.
Incubate overnight at 25°C with continuous light and shaking.
This material is now termed “lysate”.
Centrifuge the lysate in the Sorvall GSA rotor at 3,500 rpm, 10 min, 5°C.
Decant the supernatants to clean bottles.
Centrifuge the supernatant on the Sorvall GSA rotor at 10,000 rpm, 60 min, 5°C.
Decant the supernatant to clean flasks.
Add d-H2O to a final volume of 1000 mL.
Place at 4°C.
Add, at 4°C: 2-ME to 2 mM EDTA to 5 mM NaN3 to 200 mg/liter (NH4)2SO4 to 40% saturation Adjust the pH to 7.0 with NaOH.
Cover and incubate at 4°C overnight.
Centrifuge the material in the Sorvall GSA rotor at 10,000 rpm, 60 min, 5°C.
Decant the supernatant to clean flasks.
Discard the pellets.
Add, at 4°C, (NH4)2SO4 to 65% saturation.
Adjust the pH to 7.0 with NaOH.
Cover and incubate at 4°C overnight.
Centrifuge the material in the Sorvall GSA rotor at 10,000 rpm, 10 min, 5°C.
Aspirate off the supernatant.
Save the pellet.
Resuspend the pellet with 10.0 mL of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM 2-ME + 2 mg NaN3.
Centrifuge the material in the Sorvall SS34 rotor at 15,000 rpm, 60 min, 5°C.
Save the supernatant.
Dialyze the supernatant for 48-72 hours at 4°C against 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM 2-ME, 200 mg/liter NaN3.
Change the buffer once every 24 hours.
Remove the material from the dialysis.
Centrifuge the material in the Sorvall SS34 rotor at 13,000 rpm, 60 min, 5°C.
Aliquot the supernatant into microfuge tubes and store at -20°C.
Assay the supernatant fraction for lysin activity.
Save the supernatant.
