protoplasts of gram positive bacteria ?

John Ireland jawi at umich.edu
Tue Nov 30 13:22:15 EST 1999


Protoplasting of Gram positive bacteria is always a little tricky, outside
of the well studied species.  I admit that I do not have any experience
protoplasting Listeria, but I do a lot of work on certain Bacillus sp. which
are refractory to lysozyme (due to altered cell wall chemistry) and here are
a few tricks to try.

1.  Lysostaphin (as you mentioned, it is expensive)
2.  1 mg/ml penicillin G (or ampicillin will work) with the lysozyme, to
weaken the cell wall while the enzyme works.
3.  Try the ampicillin trick with lysostaphin (assuming straight lysostaphin
doesn't work).
4.  Achromopeptidase (again, expensive) about 300 units
5.  Tunicamycin (not cheap) 0.005 mg/ml
6.  mutanolysin (N-acetylmuramidase SG, Sigma), this is expensive, but works
well... the only problem is, at least with my bug, you have to get it
shipped overnight and use it within a few days (even with storage at -20) or
the activity is significantly compromised.

I wish you luck with the research,
John
jawi at umich.edu

Uwe Kaerst wrote:

> Hi,
>
> we tried to make protoplasts of listeria using egg white lysozyme - that
> worked, but took at least 16 hours which is much too long - one or two
> hours are required.
>
> After some searching I found that Sigma offers lysostaphin, a muramidase
> from Staphylococcus - that might help, but it is very expensive. Is there
> any experience with this enzyme? Or are others available for this purpose
> with much better performance than egg white lysozyme?
>
> Thanks a lot
>
> Uwe
>
> --
> Uwe Kaerst, Ph.D.
> Dept. Cell Biology
> GBF  - Gesellschaft fuer Biotechnologische Forschung
> Mascheroder Weg 1
> D-38124 Braunschweig, Germany
>
> Tel:  +(49) 531 6181318
> Fax: +(49) 531 2612313
>
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