On Fri, 20 Feb 2015 16:07:18 +0100, Pietro Roversi
<pietro.rove...@bioch.ox.ac.uk> wrote:
Dear Enrico,
I wonder if trying different protein:precipitant ratio is also a valid
strategy to crystallise very soluble proteins.
Please let me know if my reasoning is flawed and if so why!
Yes and No.
I you are using salts and your protein is still soluble when the salt is
saturated. You have an excellent
way of concentrating the protein. You will then need a co_precipitant,
like dioxane, to push it over the
edge.
With PEG this is an option for refinement, but not for serious protein
concentration.
PEG has become the most often used precipipitant, is because it work by
volume exclusion,
leaving the protein less space and effectively concentrating the protein.
http://dx.doi.org/10.1016/S0022-2836(75)80107-0
In terms of being able to extract water from the drop it is less effective
than
salts, glycerol ... etc.
So with PEG it will not work as you would think. What I do is to use
booster solutions.
In the boster solution you may have: 5M NaCl and if you want to change the
pH because
you know that acidification can increase precipitation, you also have
acetic acid in your boost.
An example is given in:
Ciccone L., Tepshi L., Nencetti, S. & Stura E.A. (2015) Transthyretin
complexes with
curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent
mixtures New Biotech. 32:54–64
http://dx.doi.org/10.1016/j.nbt.2014.09.002
You can concentrate and crystallize a very soluble protein by starting
with a low concentration high MW PEG
and by sitting drop vapour diffusion you do several boosts untill you get
a precipitate.
When you get the precipitate, you do not know where you are in
crystallization space!
But you can do precipitate transfers to other drops. The speed at which
the precipitate resolubilizes
allows you to work out approximately where you are.
Complicated, yes I agree, but it uses very very little protein.
Enrico.
Let [Prot]_0 and [ML]_0 be the initial concentrations of protein and
mother liquor solutions, mixed in volumes V0_prot and V0_ML,
respectively, to form the initial drop of volume (V0_prot+V0_ML)
In the following, let us assume that the vapour diffusion process
proceeds based on chemical potential of the ML and let us talk of
concentrations instead of chemical potentials.
The vapour diffusion process will stop when the concentration of ML in
the drop is equal to the one it has in the mother liquor, so that the
drop will shrink till its volume at equilibrium is V0_ML, i.e. the
volume of ML that was used to make the drop initially.
The final concentration of protein is therefore:
[Prot]eq = ([Prot]0*V0_prot) / V0_ML
and the concentration factor [Prot]eq/[Prot]0 is:
[Prot]eq / [Prot]0 = V0_prot / V0_ML
which show that by increasing V0_prot / V0_ML one can concentrate the
protein as much as one wants.
Please let me know if my reasoning is flawed and if so why!
Best regards
Pietro
Sent from my Desktop
Dr. Pietro Roversi
Oxford University Biochemistry Department - Glycobiology Division
South Parks Road
Oxford OX1 3QU England - UK
Tel. 0044 1865 275339
________________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Enrico
Stura [est...@cea.fr]
Sent: 20 February 2015 14:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to reduce protein solubility
Ursula,
Most compounds used for cryosolutions glycerol, ethylene glycol, propane
diol increase protein solubility.
A warning, these compounds are also hygroscopic, you need to change your
vapour diffusion methodology.
Vera L., Czarny B., Georgiadis D., Dive V., Stura E.A. (2011) Practical
Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11:
2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m
The lack of success in crystallizing proteins in glycerol are due to the
reasons decribed in the paper.
Enrico.
On Fri, 20 Feb 2015 00:33:48 +0100, Ursula Schulze-Gahmen
<uschulze-gah...@lbl.gov> wrote:
Hi Enrico,
How are you? I see you are now in France.
I have also a question about protein complex solubility. I have a
multi-protein complex that also binds RNA. This Protein-RNA complex can
be
concentrated to 5- 10 mg/ml, but starts precipitating after storage at 4
degrees for several hours ( and can often be resolubilized at room
temperature). The current buffer is 20 mM HEPES 7.3, 0.2 M NaCl, 0.05 M
KCl, 3 mM MgCl2, TCEP. I don't want to increase salt concentration. What
are your suggestions to try to improve the solubility?
Best
Ursula
On Tue, Feb 17, 2015 at 2:00 AM, Enrico Stura <est...@cea.fr> wrote:
Francesca,
The most common failure is to have an excessive amount of salt (salting
in/ salting out), glycerol or other solubilizing
ingredient in your protein solution. I would suggest that you change
the
pH and reduce the salt in your protein solution,
by microdialysis if you do not have much protein, and screen again.
If share with ccp4bb the exact formulation of your protein solution you
might get more suggestions.
Enrico.
On Tue, 17 Feb 2015 05:23:05 +0100, Mattiroli,Francesca <
francesca.mattir...@colostate.edu> wrote:
Hi all,
I am struggling with a protein complex that is too soluble. I have
reached about 20 mg/ml but I still observe very little precipitation
(clear
drops in 90-95% of the tested conditions). The proteins are expressed
in
insect cells and going to higher concentration is not easily
achievable.
I have tried different buffer conditions (salt concentration and pH)
and
I am testing temperatures. I am at a loss with what to try next.
Do you think PTMs (phosphorylation, acetylation) might be causing
this?
Any input on how to decrease solubility?
Thank you very much in advance,
Francesca
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449
Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&;
sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90
71
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90
71
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
