Dear ccp4bb,
1. Why would you need a precipitant to crystallize a protein?
The principle of salting-in/salting-out means that as you remove the salt
that
keeps a protein soluble, the protein will tend to come out of solution and
given
the right conditions, crystallize.
2. From the solubility curve as you increase protein concentration, you
will reach a point at which
your protein will become supersaturated and possibly crystallize. You just
evaporate the water.
3. Protein solubility is temperature dependent:
Huang M., Syed R., Stura E.A., Stone M.J., Stefanko R.S., Ruf W.,
Edgington T.S. & Wilson I.A. (1998) The mechanism of an inhibitory
antibody on TF-initiated blood coagulation revealed by the crystal
structures of human tissue factor, Fab 5G9 and TF.5G9 complex. J Mol Biol.
275:873-894.
Crystallized in the NMR tube when it was stored in the fridge
De Halleux S., Stura E.A., VanderElst L., Carlier V., Jacquemin M. &
Saint-Remy J.-M. (2006) Three-dimensional structure and IgE-binding
properties of mature fully active Der p 1, a clinically relevant major
allergen. J. Allergy Clin. Immunol. 11: 571-576.
The unglycosylated protein has low solubility - Concentration to 3.5 mg/ml
and waiting for it to crystallize in the fridge.
Stura E.A., Visse R., Vera L., Cuniasse P., Dive V. & Nagase H. (2013)
Crystals structure of full-length human collagenase 3 (MMP-13) with
peptides in the active site defines exosites in the catalytic domain.
FASEB J. 27:4395-4405.
A bridging peptide and low temperature induces crystallization - No
precipitant.
4. A precipitant is an agent that is used by protein crystallogenists to
avoid using too much protein
in the experiment. If salting-out does not work, PEG is used as a common
crowding-out agent.
Ideally without a precipitant your protein will be in its most pure state.
5. Maïga A., Vera L., Marchetti C., Lorphelin A., Bellanger L., Mourier
G., Servent D., Gilles N. & Stura E.A. (2013) Crystallization of
recombinant Green mamba ρ-Da1a toxin during lyophilization procedure and
its structure determination. Acta Cryst. F 69: 704-709
What about crystallization during lyophilization?
If you check throughout the literature, you will finnd many more examples.
Enrico.
--
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
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
