Variations of lysin purification
Variations of lysin purification

Protocol is to pellet lysate in the ultracentrifuge (25,000RPM, 30 min), wash pellet with 0.1M LiCl, pellet again in the ultracentrifuge, take up pellet in a small buffer volume with higher LiCl, incubate at 37C and then pellet again in the untracentrifuge. Supernatant is "purified (salt extracted) lysin".

In the samples below the salt dissociation step (small volume) was varied.

Samples 6 and 7 are essentially sample 2 except for an initial low speed centrifugation (4k, 4 min) to divide it into a cell enriched, pellet, fraction (6) and a virus enriched, supernatant, fraction (7). Together 6 and 7 should add up to 2.

Lysin assays of all samples gave similar activities except for fraction 6 (reduced activity)

Protein patterns of all lysin preps (except 6) are surprisingly similar

Adding protamine sulfate directly to the salt wash (sample 3) clears out junk (should become standard)

0.3M LiCl is adequate to release lysin

Lysin tolerates pH 4.5 which appears to give a slighly cleaner prep

Lysin activities of samples 6 and 7 were compared in detail:

The bulk of salt released lysin (perhaps all) reflects extraction from virus, not extraction from walls