Hi, everybody,
I try to crystallize one membrane protein.
All crystals were grown by handing-drop vapor diffusion at 20 degree. A protein
solution containing about 8-10mg/ml protein in 20mM Tris (pH7.5),
0.017% DDM, 100mM NaCl, 10% glycerol, 2mM DDT was mixed with an equal volume of
a reservoir solution containing 45% PEG200, 0.1 M phosphate/citrate (pH4.2).
First crystal appeared in the drop within 4 days. And one week a lot of
crystals appeared in the drops.
Our question is all of these crystals
are too small to check them by X-ray diffraction and SDS-PAGE. We
are not sure they are protein crystals or salt crystals. Our condition
seems difficult to produce salt crystal. But I am a little warry because we
use reloaded our sample to small Ni-resin column to reduce the concentration of
detergent. Maybe some nickel ion dropped off, and then our protein
sample contained some this ion. And nickel ion may react with phosphate,
and then produced nickel phosphate crystal. Could somebody tell me if
it is possible?
I attach some photos of our crystals. Could
somebody give me some suggestions about how to optimize this type crystal to get
bigger crystal?
Thanks a lot!
Yibin
2010-11-16
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发件人: Laurie Betts
发送时间: 2010-11-16 17:13:32
收件人: CCP4BB
抄送:
主题: [ccp4bb] expression of Cys-rich small
protein
All -
We are trying to express for structural studies a
257 AA eukaryotic intracellular (also possibly nuclear) protein (predicted to be
single domain all-helical) that has 12 Cysteines. No known metal-binding
function not that it couldn't happen. So far (E. coli) it expressed
solubly as MBP fusion (with an N-terminal region deleted predicted disordered)
until cleavage of MBP, then it's not soluble, including detergents added.
THe MBP fusion is usually soluble aggregate so we assume that our part is not
folded right. We have so far assumed it needs a lot of reducing agent (5
mM DTT or TCEP). Thinking of trying chaperones and insect
cells next.
Any experience out there that might help? Mostly I
wonder about all the cysteines. Don't really know if that is the
problem.
Laurie Betts