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IMPACT kit from new england biolabs...

Ted Michelini tedm at darkwing.uoregon.edu
Tue Oct 8 19:16:49 EST 1996

In article <tV+KjUAGrNWyEw15 at genesys.demon.co.uk>, "Dr. Duncan Clark"
<duncan at genesys.demon.co.uk> wrote:

> In article <siyer-0510961822540001 at bmg146.cmc.uab.edu>, Arioch
> <siyer at bmg.bhs.uab.edu> writes
> >hey all,
> >        just saw this in the september 6th issue of cell.  this is a new
> >kit from NEB that claims that you can get pure NATIVE protein (no fusions)
> >in "one" step.  basically they use proteins called inteins that are fused
> >to YPOI.  this undergoes directed cleavage when you add DTT (cleave a S-S
> >bond somewhere right before the fused region) and you get native protein
> >with no fusion domain to it.  anybody try this and if so, what is the
> >yield like??  how good is this system?  seems like a cool idea, but as
> >alwayz with any kit, i'm sceptical...any info on this would be
> >apprciated...thanx much...
> They are using a modified intein and from data I saw presented at a
> conference a few weeks back it is a very promising system. My only
> queries are:
> 1.      Is if the affinity column is reusable?
> 2.      How expensive is the affinity column 'cos it's OK purifying
> proteins on a shake flask scale but I need to work on a much larger
> scale.
> Duncan   

   I too think this is neato stuff, the acrobat file from NEB's web site
contains the full protocol and they say the chitin beads are reusable 5
times. Since we use Ni columns roughly 10X what we're supposed too, maybe
the same math holds (?). The chitin should be cheap, although I haven't
found a alt. supplier yet, perhaps one could use roughly ground
cockroaches or crabs...The manual does state the system doesn't work well
with eukaryotic proteins and that certain flanking AA's at the amino
terminus give premature cleavage.  I'm going to give it a try and will
post my results soon. The promise of one step purification/ C terminal tag
cleavage with one or no added amino acids is exciting, I'm going to give
it a whirl.


Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu

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