Stabilizing recombinant proteins in solution?

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Wed Dec 27 13:07:07 EST 2000


Hi,
I remember some time ago there was a discussion here about the ways to
stabilize proteins in cell-free extracts and during further purification
steps. I know that glycerol in buffers can stabilize proteins a little,
sucrose may do that even better in some cases.

My question is about using arginine as a stabilizer. I read in this group
that Arg can be added to protein solutions to prevent protein precipitation,
especially during concentrating (e.g., in concentrating spin cartridges),
which is especially important for proteins that start to precipitate
spontaneously over a certain concentration. I would appreciate if someone
could provide some details about concentration of arginine used, what is the
mechanism of stabilizing action of this amino acid. Are there other ways
(amino acids to use, other low MW compounds, etc.) to stabilize proteins?

I work with a yeast DNA-binding protein, and express it from a pET11-based
construct with N-terminal 6His in E. coli. As soon as I elute the protein
from a cobalt resin, I see precipitation (seen as an increasing cloudiness
in the fractions), which appears to contain my protein of interest (as
checked by spinning off the precipitate and SDS-PAGEing the pellet). Same
cloudiness can be observed in cell-free extracts if kept for too long before
applying onto IMAC column. I suspect that instability of the protein may be
attributed to it's DNA binding, since I have to digest DNA during
preparation of cell-free extract to decrease viscosity of the homogenate.
Moreover, I need to purify the protein to near homogeneity for further
crystallization needs, and if I do not digest DNA, I may co-purify other DNA
binders that share DNA strands with my protein, as well as DNA contamination
could impair crystallization in the future.

Any suggestions or comments would be greatly appreciated.
Thank you.

-Emir








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