tk at dcb-glostrup.dk
Thu Jun 10 05:02:19 EST 1999
I don't know what kind of proteins you are working with, but some of the
degradation depends on the N-terminal part of the fused protein. Meaning
that if you've got an N-terminal, which isn't tightly coiled up or bound
to the rest of the protein with disulphide bridges, it becomes more
susceptibel for proteolytic digest. It is hard to avoid this
degradation, but to some instance a lowering of the temperature will do
it, to 30 or 27 degrees celsius.
If you lower the concentration of the inducer and instead induce twice
within an hour, you may get better results as well. Nothing guaranteed
Addition of more inhibitors like Aprotinin, pepstatin and leupeptin may
help. Regarding sonication, it is probably the best method, though I
know a lot of people will dispute this. But everything depends on your
sonication protocol. Some of your protein may stay in the pellet after
sonication, in which case you'll have to revise your protocol or you may
denature the whole thing.
I'm not sure the MBP-fusion system will solve the degradation issue as
your problem does not seem to have anything to do with the
fusionprotein, GST, itself!
PS What cell line are you using? BL21, DH5alpha, ....?
Robert, Francis skrev:
> Hi everyone,
> I'm working with GST-fusion system. For two of my fusion proteins I get
> degradation of the fused part only while for others, ther is no degradation
> at all. This means that the only protein I can purify is the GST alone
> since it has a better affinity for the column than the fusion protein. I'm
> not sure, but I think I see on a coomassie-stained gel that the degradation
> occurs fisrt in the cells and continues during the course of the
> purification. I use PMSF and Benzamidine. Should I use more inhibitors?
> The lysis method implies sonication, should I use other lysis methods?
> Is there any magical methods that would prevent degradation like reducing
> the growth temperature or the inducing time? Would the MBP-fusion system be
> a better system in case of degradation.
> Francis R.
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