Maltose binding protein fusions...

Joseph Boothe jbooth at ACS.UCALGARY.CA
Thu Mar 14 10:12:08 EST 1996


Hi Steve,

I have used the pMAL-C vector to express a 65 aa protein in DH5 alpha. I 
was also unable to bind the protein to the amylose resin, but the 
recombinant portion appeared full length (determined by mass spec) after 
purification through anion exchange chromatography.

Joe

On 13 Mar 1996, Steve wrote:

> 
> Hi!
> 
> I was just wondering whether anybody out there has any experience
> with expressing fairly short (70aa) eucaryotic polypeptides as fusions
> of maltose binding protein (MBP) using the pMAL-C2 vector system (NEB).
> I'd particularly like to know which E.coli hosts offer good levels of
> stability against proteolysis without overly compromising on yield (do
> Lon-deficient strains make a huge difference? I'm currently using my usual
> cloning strain, XL1-Blue MRF' which gives excellent overexpression but
> in vivo proteolysis looks to be quite severe...). Also,has anybody
> produced modestly sized fusions that refuse to bind effectively to the
> amylose affinity column? I think I have....
> Any advice greatfully appreciated,
> Steve.
>  
> 
> 



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