Protein expression problems (very hard to find an aswer)

Mizar gianluca.molla-nospam- at unimi.it
Fri Sep 1 08:02:02 EST 2000


Dear colleague, I have a very intriguing question for you.
Well the story is very simple.
We built 4 years ago a good expression system in E. coli (BL21 DE3 pLys)
based
on the pT7.7 vector and it worked well for a long time.

Now it seems not to work as well as it did (I have about 1/10 of initial
expression).
I'm going mad to try the answer. Someone have an idea?
The cells are broken by French Press
The expression level is checked by activity assay

In order to save your time I'll tell you something about my trials:

the protein is moderately toxic for the cell (but it was never a problem
before)
I tried all induction conditions (logaritmic, exponential fase, etc)
Changed Ampicillin, LB broth, Water.
Re-transformed cells with original DNA
Re-subcloned cDNA into the expression vector
Purchased new BL21 DE3 pLys Cells
Re-checked solutions used for enzymatic assay

What can be!!!!!!!
Help me please.

Gianluca Molla PhD
Università Insubria (Italy)
gianluca.molla at unimi.it






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