Were the crystals which run different on gels from the same drop, or
separate drops?
Yes, it is possible that the protein is being cleaved in the crystal
(self-cleavage?); but it may also be that it is being cleaved in the
mother liquor, and that crystallization is enriching one form or
another. Remember that crystallization is a useful purification technique.
There should be enough protein in a crystal to get some mass spec data,
which will tell you the size of the components, and which should be
precise enough to tell you the cleavage site. This will tell you what
type of protease you are dealing with.
Then as possible remedies:
You could include an appropriate protease inhibitor during purification
to limit degradation.
You could add a bit of protease to encourage proteolysis to go to
completion. You want your sample to be homogeneous, so whether this is
useful depends on whether the cleaved part is the interesting part.
You could engineer the gene with a stop codon at or near the cleavage
site. This is what we did with HO-1, with some success.
DOI: 10.1002/pro.5560070820
You could engineer the cleavage site to eliminate cleavage.
Cheers,
On 01/16/13 06:14, LISA wrote:
> Hi All,
> I have an 36KD protein which can be crystallize in two days. Most of
> the crystals are very big. But all cystals have poor resolution,lower
> than 3.8 A. I picked some crystals, washed them in the mother solution
> and then run SDS-PAGE. It is surprised to find that different cystals
> have different components. Some crystals have several small bands
> below the band of the protein. And in some crysals the bigger size
> band (as the construct should be) almost disappared and have smear.
> Does the protein was degradated in the crystals? Did someone met the
> similar problem as I? Thanks
>
> All the best
> lisa
--
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All Things Serve the Beam
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David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
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