E.coli vector with 6His to be removed cleanly?
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Jan 15 11:39:34 EST 2001
"Sveta" <xeaa at usa.net> wrote:
:I would greatly appreciate if someone could recommend me an E. coli
:expression vector that would allow cloning to produce a 6His protein so that
:6His epitope could be cleanly cut off the protein by a specific protease.
:Any other epitope could be OK, the main concern is that no foreign amino
:acids will be introduced into the protein I want to express (it is a DNA
:binding protein, so I am worried about 6His affecting its properties).
IMHO, cutting with proteases is pretty inefficient way to go. It
only works acceptably in minority of cases and is more trouble
than it is worth in difficult cases. I'd recommend Intein-based
system such as one sold by NEB. In brief, you express you
protein as a fusion with chimera consisting of chitin binding
domain and intein, a yeast self-cleavable protease. You bind
the protein to chitin sorbent and elute with high concentrations
of DTT which activate the protease. It cleaves itself releasing
you protein from the column. Easy, cheap and quite specific
so that the purity is quite high.
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