Yeast lysis methods

MURCOTT at BSA.BRISTOL.AC.UK MURCOTT at BSA.BRISTOL.AC.UK
Mon Jan 13 08:30:00 EST 1992


I spent a long time trying to work out suitable methods for yeast lysis
for my PhD thesis which involved over-expression and mutagenesis of pyruvate
kinase in yeast.  The conclusion I came to was that mechanical methods were
considerably more effective than autolytic methods (where the yeast endogenous
lytic enzymes are liberated and used to break down the cell wall).  The main
reason for this is that yeast (I work on Saccharomyces cerevisiae) contain a
large number of proteases and in order to extract intact protein from them
exposure to these proteases needs to be minimised.  Mechanical methods of lysis
are quicker than autolytic (typically one hour as opposed to overnight) and
so reduce proteolysis time.  I strongly suggest that whatever you do you use
a powerful cocktail of protease inhibitors to further minimise proteolytic
degradation of proteins, if it is proteins you are working on.  This problem
is well reviewed by Pringle in Methods in Cell Biology (1974) vol 12, pp. 149-
184.

If you are feeling rich then I suggest that you buy a Bead-Beater which is
a glass vessel you fill with a slurry of yeast and glass beads which is then
violently stirred by a teflon impeller and is a very efficient way of breaking
open yeast.  It can cope with a range of volumes and there are not the losses
experienced with a French press.  I do not have the details of where to get
hold of this (perhaps some other netter can help?) but it is advertised
fairly regularly in the major journals such as Nature.

For about a tenth of the cost of a bead-beater I kproduced my own home made
version which is very effective and ammenable to being scaled up to very
large volumes.  Details of this and the methodology, including a further
discussion on the problem of proteases, is given in Murcott et al. (1991)
Eur. J. Biochem vol 198, pp. 513-519.
Essentially the lysis is performed in a round bottom thick walled glass
vessel into which a thick slurry of glass beads and yeast is put.  This
is then stirred from overhead with a glass paddle mounted in a commercial
variable speed power drill.  The power drill is considerably cheaper than any
equivalent laboratory equipment and much quicker to get hold of!  The glass
vessel was made in the University glass workshop, the one I use is about 8 cm
in diameter and 20 cm deep and will lyse up to 50g of yeast in one go.  The
paddle is a 5mm thick glass disk, diameter 2cm, fused to a 5mm glass rod for
mounting in the drill.  The slurry is made with the minimum of strong buffer
(teh pH will drop dramatically during lysis) and a cocktail of protease
inhibitors and glass beads are added until there is no meniscus left which
will about triple the volume of the original slurry.  This is then stirred as
fast as you can face for about an hour after which the beads are filtered
through a glass sinter and washed with a few volumes of suitable buffer.

Points to note.  The lysis vessel can be suspended in a water bath to maintain
a constant temperature, the vessel will heat up a little during lysis.  The
yeassts slurry will block the glass sinter used for filtering so a good clean
is essential between each run and mild vacuum (any frothing will denature
proteins) will sometimes aid the filtrationand washing process.  Teh glass
beads can be recycled by washing with conc HCl and then rinsing to neutrality
with water and then distilled water.

I hope this helps and good luck.


Toby


Toby Murcott		Murcott at uk.ac.bristol.bsa

Dept of Biochemistry
School of Medical Sciences
University of Bristol
University Walk
Bristol  BS8 1TD
UK



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