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 .
