Another point not yet brought up is how do you express ?
things to check:
- different media (LB. TB, auto)
- when do you induce (mid log phase, or end log phase)
- how long do you induce (before reaching stationary phase, going over
night into starvation phase)
- how much IPTG do you add
- MBP purification, despite not being in the manual I noticed higher
recovery rates when not following the protocol and using buffers ~ pH 8.5
- when you run your sizing how concentrated is your sample ?
- do you have divalent ions in your buffer e.g. Mg or if you have them
around, remove them by addition of EDTA or EGTA
- glycerol in your sample buffer as chemical chaperone ? 10-20% would do
it perhaps.
So I would say, don't throw your construct away, unless you have tried
all the other options first.
Do you have a CD available, you could also check your sample if it's
spaghetti or not.
Good luck,
Juergen
Daniel Jin wrote:
Hi,
I have been trying to express a rat protein in bacteria. The
MBP-fusion expressed at very high level (~ 40 mg/L) while the
GST-fusion and His-tag only gave inclusion bodies. The problem is that
all protein runs in the void volume on a size-exclusion column (s-200,
hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or
cleaved sample. There is no Cys on this protein so there is unlikely
any disulfide bond related problem. Anything I can do before I throw
away this construct and try insect or mammalian cells? Thanks.
Best,
Chen
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