E.coli expression sysytems

iwilson at molbiol.ox.ac.uk iwilson at molbiol.ox.ac.uk
Wed Nov 3 07:53:36 EST 1993


In article <1993Oct29.185428.1 at opal.tufts.edu>, skrishna at opal.tufts.edu writes:
> > [...] 
> 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)
> 

Dear Sanjay,
	With an His6-EPO construct, one of our students found a protein, which 
was not her construct, stciking to the Ni-column. She was using a BL21 strain 
as well. Could you say what size this otehr protein was, so that we can 
compare results?

Iain Wilson
Dyson Perrins Laboratory, University of Oxford
iwilson at molbiol.ox.ac.uk



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