piero.morandini at unimi.it
Fri Nov 26 11:40:08 EST 1999
I am expressing several proteins as GST or His-tag fusion
and I am encoutering a few problems:
1) What is the most effective cocktail to stop proteolysis
in E. coli after cell disruption?
I am using "Complete" (from Boehringer) + PMSF, but they do not
seem to be enough.
2 Has anyone tried to add protease inhibitors during/at the end
of the induction? (so that one is able to block also in vivo
3 Concerning thrombin or factor Xa, has anyone thought of making
a GST or His tag version of the enzyme, so to be able to remove it
(or to purity it easily) after reaction.
(I am of course thinking of using the his-tag version of thrombin
to cut the fusions with GST.
4 Do people remove the thrombin with gel filtration or use the
You can address answers (if any) directly to me. I'll make the a summary
for all the people
Thanks in advance
dept. of Biology
Univ. of Milan
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