Stabilizing recombinant proteins in solution?

[Frans]

Hi,
I can't give you the exact concentration of Arg. I'v read it somewhere and I
think it's 3M.
To keep the protein in solution may be you can use detergents like GuHCl
(6M) or Urea (8M). The protein then denaturates but after purification you
can dialyse the detergent out of the solution. Do this slowly so the protein
can refold to its natural structure. If the protein still precipitates you
can try to make a construct with a fragment of the protein. We had good
results with a humane protein fragment (see Perez JM et. al., Protein Expr
Purif 1998 Jul;13(2)259-267; Perez JM et. al. Structure Fold Des 1999 Feb
15;7(2):217-226). You can find an abstract in PubMed at
www.ncbi.nlm.nih.gov.
On SDS-PAGEing the isolate was pure, activity was good and the concentration
was high.

Good luck,
Frans
(biochemical technician)


"Emir" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote in message
news:Ccq26.6$x3.172 at uchinews...
> 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
>
>
>