Hello Sabine,
Maybe this is an option:
Structure, Vol 14, 1617-1622, November 2006
Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization
best regards & good luck
Christian
Schneider Sabine 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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