Hello Everyone! We're having some trouble with our protein expression
system. Like almost everyone else, we use an expression vector to express
a His-Tagged fusion protein, which we subsequently purify over a Qiagen
Ni-NTA column. What we've noticed is that lately our proteins are
beginning to show up in uninduced bacteria. The tac promoter is supposed
to drive transcription, and shouldn't be active in the absence of IPTG.
Why are our proteins expressing in uninduced bacteria?
First of all, we do not have any IPTG contamination in our glass flasks,
which are autoclaved prior to each round of preparative bacterial growth.
And yes, we know about the so-called pLys variant of BL21 cells which
really represses the expression of T7 polymerase, thus giving almost no
expression of the his-tagged fusion protein until IPTG is added. And No,
we're not using the pLys variant, and maybe we should be....
But I think we have a more fundamental problem. I think our bacteria are
changing somehow so that expression becomes constitutive. This is also a
problem because we can't try various levels of induction in an attempt to
reduce subunit aggregation. Of course, the insoluble proteins are much
harder to purify using Gu-HCL or Urea...
If anyone has had this experience and knows why it happens, I would sure
appreciate an email or a post!!
Thanks in advance...