ekhatipo at NOSPAMmidway.uchicago.edu
Thu Sep 21 13:06:49 EST 2000
If you are using a commercial vector you will most probably get a spacer btw
6his and your gene, even if your cloning strategy allows removing maximum of
the MCS. In my experience I did have success both with commercial vectors,
and when I introduced 6his by PCR immediately up- or downstream of the gene
of interest. However, I know from other people that it is all totally
empirical: you cannot predict that your protein will or will not bind metal
affinity resin, although in most cases it will, and in general the
introduction of a spaces would facilitate the binding. The biggest concern
to me is to prove that the modified protein (6his + spacer) is functional in
vivo, which can be tested by expression of the protein under its natural
promoter in a null-mutant to check whether the expression complements the
function. But the necessity of the latter certainly depends on your aims.
"Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
news:Pine.SGI.3.96.1000921153325.6194204C-100000 at mole.bio.cam.ac.uk...
> What is the experience of people using hexahis tags for purification? Do
> I need a linker to separate my protein from the tag (like a string of
> glycines), or should I just remove the stop codon and add 6 his (or is 10
> better?). Mike.
More information about the Methods