low level expression vectors

Randy Willis willis at gandalf.psf.sickkids.on.ca
Fri Apr 11 15:06:40 EST 1997


Koen A.L. De Smet wrote:
> Either these proteins prefer being expressed in mycobacteria, or the overexpression makes them insoluble in E.coli.


Dr. De Smet,

I would almost bet that both explanations are partially correct and I do
not think that they are all that exclusive of one another.  It should
come as no surprise to anyone that when you greatly over-express any
protein in an alien environment, the host cell will deal with the
intruder in any way possible.  We have often seen cases where proteins
or domains of proteins from other systems will be insoluble when
over-expressed in most coli strains.  Sometimes to get around this, we
have grown and induced the bacteria at lower temperatures and this has
resulted in the cells having more time and the ability to deal with the
alien protein such that the protein produced was more soluble and the
culture reached a higher final OD such that we ended up with more total
protein per unit culture volume (and when you're doing experiments in
isotopically-labeled media, this can be critical).  I think that this is
also why the OD of induction can also be critical for an experiment as
most systems seem to prefer to be induced in log phase (although this
must be tested for each system and cannot be taken as an absolute).  It
may sound strange to think of your culture as having its own Zen or
Karma, but I have often found that a happy bug is a productive bug.

Please let me know how your experiments go.  I would be greatly
interested in seeing this phenomenon studied further and more
carefully.  Alas, the time constraints of working for others.

Good luck,

Randall C Willis, Researcher
Biochemistry Research, Hosp for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)

willis at gandalf.psf.sickkids.on.ca



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