there are two accepted ways to break a yeast cell wall, physically or
enzymatically.
On a small scale most people use a french press to physically disrupt yeast
or an enzyme called zymolyase to make spheroplasts from yeast before
proceeding as per E. coli, usually osmotic rupture with sonication. Both
work fine, the caveat being "for small volumes", the french press is quite
low flowrates (depending on its size) and the zymolyase is expensive (like
most good enzymes).
If you refer to Mitchelhill et. al., J. Biol. Chem. 269, 4, 2361-2364, 1994
you will see the method I used to obtain an extract from up to 500g of
yeast at a time to prepare useful amounts of a yeast kinase called snf1.
I purchased my yeast fresh from the local bakers suppliers the day before I
wanted to do the preparation then used a Waring blender, glass beads and
liquid nitrogen to make "yeast powder" which was "warmed" to -70 degrees
overnight. The powder was extracted using a conventional extraction buffer
the following morning. The method generates a thick soup containing vast
amounts of protein and a fair bit of lipid which I reduced with
precipitations and a batch ion exchange step before more specific
purification methods.
The advantage of this approach is the amount of protein that is recovered
and the fact that, during the powder preparation, the extract never gets
above the temperature of liquid nitrogen, a pretty good method of limiting
proteolysis I would have thought.
Hope this helps, I can fax you a protocol if you like.
Regards....Ken
>We would like to prepare a total protein extract from S. cerevisiae. I
>am trying to find what lysis buffer and procedure to use to get efficient
>protein extraction (including membrane proteins).
>
>Thanks,
>
>Chris (turck@itsa.ucsf.edu)
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Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.medstv.unimelb.edu.au/abrf.html
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