GST/His Tag expression woes
atutter at aim.salk.edu
Wed Mar 8 13:43:50 EST 2000
> I have not varied the length of induction, nor the concentration of
> IPTG. Can anyone
> out there offer any suggestions as to which conditions to vary. Is this
> doomed to fail?
> Should I start looking at baculovirus or gateway technology?
you just suggested the right things to do. it sounds like the fusion is
toxic to the cells at the current induction conditions, which explains why
you see a faint band around the size of gst-- it is expressing weakly and
getting degraded back down to gst--this is common. try milder induction
conditions, like 0.1-0.2mM IPTG instead of 1mM. I routinely use 0.1mM for
all my gst fusions. 3 hour induction is a good starting point, i doubt
varying that much will help, but you may try one and two hour inductions. i
have never used the strains you indicated, so i can't comment on them. i
always use BL21 DE3. if you are using a codon-optimized strain, then you
maight be over-expressing. again, lower IPTG may help this.
Salk Institute for Biological Studies
10010 N. Torrey Pines Rd.
La Jolla, CA 92037
email: tutter at salk.edu
More information about the Proteins