10:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein expression problem
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
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
Chen and David,
Before adding detergent, be forewarned that the MPB in many fusions
will not bind to an amylose column in the presence of most
detergents, particularly maltoside detergents. It has been the bane
to us so we have engineered MBP vectors with His tags to deal with
this. Wha
I wholly agree with the below. I am not sure how well E.coli can correctly fold
snaky
misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out
that tags do it
sometimes...
"Folded by association" for insoluble proteins has often not worked well for
me. Sometimes, when it
'
> 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
Hi Chen,
You could try adding some detergent or other solubilising agent (eg
NDSBs) to your buffer.
Have you tried other pHs? If you are sat near to or on the pI of your
protein, it will be at its least soluble and more likely to aggregate.
I've had protein behave like yours at pH 7.5 but behave p
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
