Fusion protein toxic to E. coli.?

Randy Willis willis at gandalf.psf.sickkids.on.ca
Fri May 30 10:11:06 EST 1997


Zhonglin Chai wrote:
> I did observe that the E.coli. cell density dropped soon after IPTG
> induction.

Zhonglin,

Toxicity of a foreign protein does occur on a regular basis in E.coli
although I must admit that this is the first time I've heard it for GST
fusions.  In the past, whenever I have come across this problem, I have
been forced to do the inductions at much lower temperatures.  I am
working on the assumption that this means that the protein is expressed
at a much lower rate which allows the cells time to deal with the alien
intruder.  The following protocol may be of assistance although you may
have to try variations of this for your system:

inoculate and grow at 37C
at OD600=0.4, change temp to 30C
at OD600=0.6, change temp to 25C
at OD600=0.8, change temp to 20C
at OD600=1.0, change temp to 15C
at OD600=1.2, induce with IPTG
grow for 8-36h at 15C, taking time points for induction and OD

(unless you work in a really cold room, I would suggest moving the
shaker into the cold room for growths at 15C)

Another possibility is to change your fusion system to something which
has a tag which promotes precipitation of your protein in the cell into
inclusion bodies.  This may assist the cell in dealing with your
protein.  The only problem, of course, is then being certain of
refolding at least enough to cleave the tag.  In the pET system from
Novagen, there is a T7 tag which can be added and this is from the T7
coat protein and promotes pptn.

Good luck and let us know how things go.
Randall C Willis, Researcher
Biochemistry Research, Hospital 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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