Protein expression systems

Johan deBoer jdboer at SOL.UVIC.CA
Thu Oct 8 00:29:03 EST 1992


Forwarded message:
> 
> We have been using the Maltose-Binding System over the last year
> with success except in one area which I think is worth mentioning.
> One can cleave the MBP from the protein of interest with factor Xa.
> One is then supposed to be able to to separate the cleaved
> MBP from the target protein by passing the cleaved mix over the amylose affinity
> column.  The protein of interest should pass through and the MBP should be retained.
> We find that after cleavage, the MBP fails to bind to the column.  I have talked
> 
We have been using this system with pretty good succes. The first problem we
had was that with the original pMAL vector both cleavage and binding to amylose
was not very good. This was solved by using instead the pIH902 vector which
puts a short amino acid linker between the MBP and your protein. This 
apparently makes it better accessible to Xa and to the amylose column. It worked
a lot better. 
The problem with binding of the MBP to amylose after cleavage (we had that 
problem too) was because we could not really get rid of the maltose. What
we eventually discovered was that simple passing the cleaved mixture over
an hydroxy apatite ultrogel column separated the MBP and our protein 
beautifully. The MBP sticks to the column and our protein (APRT) runs
through. this may not work for all proteins and you may have to fiddle 
with the salt concentrations of the HA column and the sample, but in our
case it worked very well.

Johan de Boer
jdboer at sol.uvic.ca




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