E.coli expression sysytems

iwilson at molbiol.ox.ac.uk iwilson at molbiol.ox.ac.uk
Fri Oct 29 10:06:00 EST 1993


In article <hersh-271093111242 at bromo.bocklabs.wisc.edu>, hersh at macc.wisc.edu (Rick Hershberger) writes:
> In article <1993Oct26.152415.1 at molbiol.ox.ac.uk>, iwilson at molbiol.ox.ac.uk
> wrote:
> 
>> Suggest try a His-tag pET fusion vector from Novagen (AMS Biotechnologyin UK). 
>> This is better than GST-fusion vectors in our experience.
>> 
>> Iain Wilson
> 
> I've tried GST-fusion with a particularly insoluble protein with which I am
> working, and fusion to GST didn't improve solubility or allow for affinity
> purification.  Could you describe the circumstances under which you arrived
> at your decision that the HisX6 tag system works better?  I'd like to know
> if it's worth yet another go at affinity tagging of my protein.  Thanks
> 
> Rick Hershberger

I have asked the student in our group for fuller details: she writes:

"In my experience a GST-fusion of EPO was better than His6-EPO. In the former 
case even though 90% of the protein was insoluble, the 10% of soluble protein 
contained bioactive species (~2% of the 10%). In the latter case I could not 
affinity purify anything other than a mystery non-His6-tagged protein!
However Boissel and Burn (JBC, 1993) used His10 at the C-terminus to purify 
EPO (although admittedly the protein fusion had to be refolded) which was 
active.
Talking to others at the Inst. Mol. Med. in Oxford, Amir Moghaddan used 
pGEX-2T to express active GST-PDECGF (see Biochemistry 1992, 31:12141-12146) 
and also found that pET-14b (His6) to be as good if not better. Chris Addison 
also used pET-14b to produce milligram quantities of her protein (I don't know 
which) and since the lab she works in has a pathological hatred of pGEX-2T, I 
can't compare the two systems in this case.
So to summarise, I think it's very difficult to generalise. The only solution 
is to try the systems and see which is best for the particular protein.
Also: what about considering maltose-binding protein fusions. They result in 
periplasmic expression and are supposedly very good, but I can't quote any 
satsified customers.
Ros Wilks"

Therefore, Rick, it's a very complicated issue: we have had good results in 
terms of expression with pEt-14b with a glycosyltransferase, but have produced 
low amounts of soluble bioactive EPO with pGEX. We are working on the 
purification of the His6-glycosyltransferase, so don't know whether that 
works, but the T7 promoter does!

Iain Wilson
Dyson Perrins Laboratory, University of Oxford



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