asaeed at research.neu.edu
Sat Jul 7 20:35:13 EST 2001
I am new to the yeast world and am looking for some advice regarding growth
/ manipulation of S. pombe. I am using S.pombe for protein expression
pretty much as described by Stratagene for their ESP yeast protein
expression system. I did a small scale experiment which turned out fine -
i.e my protein of interest was expressed well and was soluble and active.
The large scale attempt was not so good. My main questions are:
(1) For the large scale prep I omitted the vegetative growth phase in YES
medium and directly inocculated an EMM starter culture with cells from a
fresh plate. According to Stratagene this is OK for proteins which are not
toxic for the cells. Is this standard practice? I had a feeling that the 1L
culture was not very dense?
(2) Is there a rule of thumb for volume of inocculum to use. I was told to
use 5-10 ml of an overnight (30oC) grown EMM culture per litre?
(3) What is the average cell mass / wet pellet weight one can expect to
obtain from a litre of EMM culture grown for 18-20 at 30oC ?
(4) Does anyone know any tricks for recovering maximum amount of lysate
after bead beating? I am using the biospec mini bead beater and I have a
feeling that a lot of liquid stays trapped within the glass beads. Is there
a better alternative to bead beating?
I think this system is great because it gave me soluble active protein when
the E.coli systems I generally use utterly failed. I would appreciate any
insights/tips/pointers from anyone who is used to working with these
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