Re: [ccp4bb]: ccp4bb]: protein oil out

[Garavito <garavito@magaera.bch.msu.edu>]

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On Tue, 27 Nov 2001, hongmin LI wrote:

> hello,
>
> Sorry this question is not CCP4 related. I am working on a protein
which
> is highly soluble. I can easily concentrate it to 60mg/ml. While the
> protein was not precipitated from most chemicals (PEG, (NH4)SO4,
molonic
> acid, ...), instead, it is oil out. Does anybody have similar problem
> before? Any suggestions ? Thanks in advance.
>
> Hongmin

I was intrigued by this problem and also by some of the replies.  The
"oiling out" of protein in the presence of precipitants is a quite
common
problem.  It is also a hallmark phase phenomenon of colloidal and
polymer
chemistry.  Oiling out during the crystallization of membrane proteins
is
often a detergent-dependent phenomenon (as Ed points out), but PEG alone

does displays the same phase behavior.  It is interesting to note that
phasing out is seen in PEG/dextran sulfate mixtures, a phenomenon much
used
is some areas of biochemistry.  As Jeroen points out, some of the
interactions that give rise to this phenomenon are fairly well
understood,
although I disagree that one should always avoid being at the pI.

However, the main unresolved issue is how to deal with oiling out.  One
important point to make is NOT to consider it an oil; it is a a dense,
hydrated colloidal liquid.  The same is true for the phases rich in
membrane proteins or detergents.  Thus, adding detergents or hydrophobic

additives can be counterproductive.  Remember that the protein-rich
droplets are at VERY HIGH protein concentration (100-200 mg/ml), about
the
concentration one sees in a crystal.  The trick is then to get the
molecules to nucleate, most likely by changing the surface charge (pH)
or
hydration on the protein.

You might try looking up work by Bill Ray on phosphoglucomutase (J.
Cryst.
Growth 76, 562-76, 1986 and in JBC in that year) where he grew crystals
from a two-phase "coacervate" system.  He did an extensive study on
crystallization from a PEG/salt system with two liquid phases.  Check
into
the temperature and pH dependence of the phasing out.  By using
temperature
or pH gradients, crystallization might be induced.  This may also mean
you
might have to shift to microdialysis or free interface diffusion in
order
to allow you to change a single variable like pH.  Finally, have you
tried
concentrating your protein to 100 mg/ml and just dialysing out all salt
and
buffer at different pHs (as Bart suggested)?  This neglected method does

work for some proteins and viruses.

While no definitive answers can be given, at least we can assure you
that
this is not an uncommon phenomenon.

Michael


> Hello,
>
> especially around the isoelectric point of a protein,
> repulsive Coulomb forces decrease and attraction increases among
> the molecules. The latter can lead to phase separation, as you
> observed, resulting in one phase with low protein concentration
> and the other with very high protein concentration e.g. oil.
> The oil can be considered as a van der Waals gas.
>
> Now this is actually not so good. The only thing you can do (besides
> trying variants of the protein) is to try and crystallize it far away
> from the isoelectric point. The latter will not be easy at all if not
> impossible.
>
> My advice, try combinations of PEG and salts at pH values far away
from
> the isoelectric point.
>
> Jeroen.

--
*****************************************************************************

R. Michael Garavito, Ph.D.                   Email:  garavito@msu.edu
Dept. of Biochemistry & Molecular Biology   Office:  (517) 355-9724
Michigan State University                      Lab:  (517) 353-9125
East Lansing, MI 48824-1319                    FAX:  (517) 353-9334
*****************************************************************************

--
*****************************************************************************
R. Michael Garavito, Ph.D.                   Email:  garavito@msu.edu
Dept. of Biochemistry & Molecular Biology   Office:  (517) 355-9724
Michigan State University                      Lab:  (517) 353-9125
East Lansing, MI 48824-1319                    FAX:  (517) 353-9334
*****************************************************************************