Dear Kavya,
I'm sure there are several similar stories to learn from.
I've worked with a point-mutant of a "normal" protein which made it
thermostable - as revealed by DSF. It also resulted in an increased
solubility, and the thing could be concentrated to 100 mg/mL and left on
the bench for weeks with no alteration. It crystallized, but sadly (and
understandably) never diffracted.
One option for crystallizing your protein could be to look for buffers
which make it a bit less soluble? So you wouldn't have to concentrate it
to so high values, and start nucleation at a lower concentration? Again,
scanning several buffers/protein concentration ratios, using DSF, would
be one possibility. You're looking for the sweet spot to start
nucleation. Don't be afraid to try uncommon buffers, pH values,
temperatures.
Other option would be to test using nucleanting agent - crystallophores,
Naomi's nucleant, nanodiamonds, dried seaweed powder, hair... Or even a
crystal seed from another protein might help.
Good luck!
Deborah
On 05/02/2024 10:27, kavyashreem wrote:
Dear All,
Has anyone worked on a protein which is highly soluble even at 80mg/ml?
We have one such candidate, which does not precipitate even at 80mg/ml
instead forms phase separated globules in crystallization plate, which
eventually hardens over a period of 1 to 1.5 months (which is
florescent under UV microscope.)
We tried screening at different pH, but failed to get any hits.
Since we got few conditions in which the phase separated globules
solidified, we focused on them and expanded with 120mg/ml protein,
still there were not visible precipitates except for the phase
separation. This has been a challenging target so far. We have tried
with different constructs, which unfortunately are not soluble!
Does POMs help in such cases? Or do you have any other suggestion.
Thank you
Regards
Kavya
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Deborah Harrus, Ph.D.
PDBe Archive Project Leader, Biocuration Lead
PDBe - Protein Data Bank in Europe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
http://www.PDBe.org
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