Dear fellow
crystallographers,
I have two crystallization-related
questions, any suggestions will be greatly appreciated.
1)
We have a protein that behaves
really well during purification. Only two chromatographic steps (Ni-NTA
an gel-filtration) are needed to reach a purity of > 99%. Following
gel-filtration, the protein can be concentrated to as high as 50 mg/ml easily
in a very ordinary condition (200 mM NaCl, 20 mM Tris (pH = 8.0), 1 mM EDTA).
However, when we performed crystallization screen, we found that this
protein is extremely sensitive to the presence of any non-ionic precipitants.
It precipitates like crazy when encountered PEG (any size, any form of
derivatization), MPD
etc. Naturally we started to cut back on sample
concentration, we tried 20, 10, even as low as 5 mg/ml but it still
precipitated badly. I am pretty sure someone must have faced this
problem before, any advices for us?
2)
We are trying to soak a 14-residue
long phosphopeptide into a protein crystal. But I think the solubility
of this peptide is just too low in the crystallization buffer so we were
unable to detect its binding. We tried to add acetonitrile to improve
solubility, but apparently the protein crystal didnt like it. Again,
Id really appreciate any suggestions.
Many thanks.
Nei-Li
