Newest lysin purification protool
1. Pellet 500ml cells (5min x 5000RPM). Resuspend in 20ml MBBM, add 20ul tetracyline
2. Add 150ul 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 centrifugation at 25,000RPM (50.2Ti) for 17 min at 20C.
4. Re-suspend cells in 20ml 25mM KOH, 50mM MOPS, 2mM Na2EGTA, 0.1M LiCl(pH 7) (wash).
5. Centrifuge 25,000RPM for 17 min at 20C, discard supernatant.
6. Suspend pellet in 1.5ml 25mM KOH, 50mM MOPS (pH 7), 60ul 10M LiCl, 30ul 0.1M Na2EGTA incubate 50 min at 37C
7. Centrifuge 25,000RPM for 17 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 shows that all chlorophyll has been released and no cell wall material pellets at low speed (wall has been solubilized).
The final high speed pellet (after step 7) showed a white layer (virus) remaining in the pellet.