I have experience with some proteins that don't tolerate freeze-thawing
very well. It's hard to say exactly what the physical chemistry of this
is, but it probably relates to (1) aggregation due to high concentration
or protein or salts during the freezing process as water is removed,
and/or (2) pH shifts due to changes in pKa of buffers/proteins as the
temperature is lowered. Usually freezing in whole cells is less
problematic than freezing purified protein solutions, but there are no
absolutes. One protein we worked on could only be stabilized from cradle
to grave in 20% glycerol, 100 mM DTT, and 4 deg C. Would not tolerate
freezing, ever. Not even in cell pellets. Died at 25 deg C in a couple
of hours--had to work quickly to do kinetics. Worst...protein...to work
on...ever.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 9/29/2014 11:02 AM, Andreas Förster wrote:
Dear all,
I've encountered people who refuse to freeze cells and always lyse
fresh pellets. Better protein, they say. I've never had reason to do
so myself, or even to believe in their voodoo. Up until now, maybe.
My protein expresses well and is almost all in the soluble fraction in
an expression test from a fresh pellet. The large-scale expression
from the same pellet, now frozen and thawed, yielded 90% insoluble
protein.
If it's the freezing that dooms the protein, I'm happy to redo the
fermentor run. Are there other examples out there of this?
Thanks.
Andreas
