GST-fusion degradation!

Loïck Le Dantec thesax at club-internet.fr
Thu Jun 10 09:46:59 EST 1999


>> 
>> 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.
>> 
>> Thanks
>> 
>> Francis R.
>
>--
There's some hints inGoeddel V.(1990) Systems for heterologous gene
expression. Methods in Enzymology, 185, 3-228.
Periplasmic export may be used (MBP-fusion with the pMAL-p2 system) as
a means to remove the protein of interest from the proteases.

hth
(sorry for my english)



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