On Fri, 20 Feb 2015 16:07:18 +0100, Pietro Roversi  
<[log in to unmask]> 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 [[log in to unmask]] on behalf of Enrico  
> Stura [[log in to unmask]]
> Sent: 20 February 2015 14:36
> To: [log in to unmask]
> 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
> <[log in to unmask]> 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 <[log in to unmask]> 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 <
>>> [log in to unmask]> 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: [log in to unmask]                             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: [log in to unmask]                             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: [log in to unmask]                             Fax: 33 (0)1 69 08 90 71