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

More information about the Methods mailing list