GST-fusion system hints and questions

Randy Willis willis at gandalf.psf.sickkids.on.ca
Tue Feb 18 18:30:24 EST 1997


Bart,

I have to admit to not having had a lot of experience specifically with
the GST fusion systems, but I am willing to share what I have found
generally for protein expression.

Our lab has gotten into the habit of doing fresh transformation anytime
that we need to do an expression.  This seems to limit some of the
variability from batch to batch and eliminates the concern of bacteria
having been cured of their plasmid.

As well, in one situation where degradation by cellular proteases
(during protein expression) was a problem, I found that I could limit
the in vivo degradation by performing inductions at 15C.  To do this, I
started the culture at 37, let it double and then dropped to 30C, after
an hour or so dropped to 25C so on.  Start with a low inoculum OD and
keep in mind that this was in M9 (minimal medium).  As well, I found
that I could induce the culture at an OD600 of ~1.2 and let the
induction go overnight (or 8h) and ended up with final OD's in the range
of 2.7-3.0 which is great for minimal.

I then took care of an in vitro proteolysis with a melange of protease
inhibitors including some of those mentioned.  I tried the BM tablets
but found them too expensive for large scale work.  I was then able to
limit the protease inhibitors used in later steps of purification.

Generally, at higher temps like 37 or 30, I've found that long
inductions can lead to a higher level of inclusion body formation.

All of our work has been in BL21(DE3) cells, so I can't really comment
too much on bacterial strain variability.

In the long run though, this protein expression stuff is a total crap
shoot and one shouldn't give up too early.  I think that I'm starting to
freak out some of my co-workers as I'm talking about keeping my bugs
happy and appear to be getting into bacterial karma...but hey, whatever
works for you.

Good luck,

Randall C Willis, Researcher
Biochemistry, 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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