E.coli expression sysytems

skrishna at opal.tufts.edu skrishna at opal.tufts.edu
Fri Oct 29 18:54:28 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 am working with a group right now on the purification of a particularly
hydrophobic (integral membrane 12 helix) protein with which we have tried BOTH
fusions.  I am letting Dr. Laura McMurry, who has worked with the actual
purification, reply:
	"We made the following fusions: GST at N terminus, Mal E at N ternimus
hisx6 at C terminus.  The protein is extracted in detergent (dodecylmaltoside).
The GST fusion gave two species, neither with the C terminus of our protein.
The fusion would not stick to the affinity resin.  The MalE fusion fave about 5
species containing MalE, only one of which had the C terminus of our
protein--all purified on the affinity column and were released nicely but who
wants them.  The his fusion was reasonably successful, stuck nicely to affinity
Ni column and eluted nicely.  However in strain BL21 a second protein
copurified.  This protein was not found in E. coli K12.  
We do not know if the purified his fusion protein is native/active."

	If you have further questions on the expression system please email me
at skrishna at opal.tufts.edu and I will pass them along.
					Sincerely,
					Sanjay Krishnaswamy
					 (skrishna at opal.tufts.edu)



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