Newest lysin purification protool
Newest lysin purification protool
1. Pellet 700ml cells (4min x 5000RPM) in the F14-6x250y rotor. Resuspend in 40ml MBBM, add 40ul tetracyline
2. Add 40ul purified PBCV incubate 16hrs under fluorescent illumination with a 16 hr light/8 hr dark cycle at 25C on an orbital shaker (~95 rpm).
3. Collect cells and virus by centrifuging 25KRPM (50.2Ti, 1x3.5" polycarbonate tubes) for 20 min at 20C.
4. Re-suspend cells in 20ml 25mM KOH, 50mM MOPS, 2mM Na2EGTA, 0.1M LiCl(pH 7) (wash).
5. Centrifuge 25KRPM for 20 min at 20C, discard supernatant.
6. Suspend pellet in 3.0ml 25mM KOH, 50mM MOPS (pH 7), 100ul 10M LiCl, 50ul 0.1M Na2EGTA incubate 50 min at 37C, then mix with 100ul 1mg/ml protaminesulfate
7. Centrifuge 25KRPM for 20 min at 20C. Collect supernatant (avoid DNA)
This protocol assumes that salt extracts lysin from the virion rather than from cell walls. Yield seems to confirm this assumption.
Quality control: lysin assay of the lysate shows that all chlorophyll has been released and no cell wall material pellets at low speed (wall has been solubilized).
At step 5 viscous material (DNA) is apparent. Protamine sulfate serves to ppt the DNA.
The final high speed pellet (after step 7) showed a white layer (virus) remaining in the pellet.