Sabine,
There are protocols to modify surface residues that can help with crystallization, and make the protein less soluble. Unfortunately, I’m drawing a blank on the details. I remember someone in Andrzej Joachimiak’s group was working on this as a rescue approach for the structural genomics pipeline, and it had been previously published by others.
Have you looked at the protein with dynamic light scattering?
Are their cysteines? Are you using reducing agents? Sometimes mutating cysteines to serines can help.
How much purification have you done? Try ion exchange and gel filtration.
Do you have more than one ligand? I think there is quite a lot of variability in how different ligands promote crystallization. Are you adding the ligand in excess? Try a few different molar ratios. If if has high affinity, you might want to try removing excess unbound ligand at the end.
Good luck!
Kendall
On 2/22/07 9:08 AM, "Schneider Sabine" <[log in to unmask]> wrote:
Hi everyone,
I am trying to crystallise an extremely soluble and charged protein. It is ~30kDa and has an estimated PI of 5.2 and theoretical charge over pH range 4-10 from + 24 to -29. It is still happy at a concentration of 190mg/ml and fully reconstituted with its ligand.
I have tried high throughput crystallisation with 10 different screens from Nextal with concentrations of 60, 100 and 150mg/ml with no NaCl and NaCl concentrations of 100mM, 300mM and 1M in either Hepes pH 8 or Tris-HCl pH 7.5.
The distribution of heavy precipitation, light crystalline precipitation and clear drops through out the screens locks like I am in the right concentration range around the 100mg/ml, but I am not getting any real hit. There are some drops with extreme phase separation. I also tried changing the temperature from 20C to 4C.
I chased up a few conditions with this strong phase separation (or where I imagined little objects...) by manual screening and also adding additives like 3% Succrose, 50-200mM LiCl, 100mM EDTA, varying the PEGs (1500, 3350, 4000, 6000, 8000) as well as adding NaCl to the reservoir solution in sitting as well as hanging drop screens. But I am just getting nowhere - either just precipitation or the drop stays clear with the strong phase separation.
I also re-cloned it with chopping of a few more residues on the N-term where according to a secondary structure prediction a helix starts and it is still very happy at high concentrations, but again nothing in the high-throughput screens.
Has anyone any suggestions what else I could try?
Thanks!
Sabine
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