His-6 tagging of proteins for purification

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Sun Oct 24 21:29:27 EST 1993

dresnick at athena.mit.edu asks:
>Has anyone out there used the Hisx6 tags for purification of recomb.
>expressed proteins?  {stuff deleted}
>1) Does the His6 tag need to be at the very N or C terminus for the
>   protein for the protein to bind to the Ni2+ column?  Or can
>   it be internal as long as it is solvent exposed?
>2) Has anyone actually done this in yeast?  Would you expect it to
>   affect secretion in any way if this sequence were placed
>   immediately after a signal sequence (e.g. invertases')?
I passed the questions on to my colleague across the hall, Bob Bouchard,
who is not yet Internet-savvy.
Bob's response follows:
Your query was passed along to me by Bill Morgan.  I have been attempting
to prepare a maize small heat shock protein in quantity using the hisX6
method followed by enterokinase cleavage by cloning the cDNA into the
pTrcHis prokaryotic vector from Invitrogen.  While this is not precisely
analogous to what you intend to try, I have encountered a couple of
phenomina that may be germain:
1.      The Ni2+ column definitely works.  My particular product goes with
the insoluble fraction in the bacterial proteins, so I had to prepare and
bind it under Invitrogen's deaturing conditions.  The product appears to be
over 95% pure in the peak fraction based on both 1-D and2-D gels after
washing and eluting according to their standard procedure.  However;
2.      Cutting of the fusion protein may be a problem even if the linker
is placed optimally at the N-terminus.  My particular fusion protein has
proven quite refractory to the enterokinase cleavage under the standard
conditions for the enzyme given by Biozyme, the supplier recommended by
Invitrogen.  I am currently experimenting with digestion in various
molarities of urea (the enzyme is active up to 5M!) and different pHs
(trying to work around the pK of histidine) to see if I can solve this
problem.  When pressed, Invitrogen's technical services confess that every
protein is a unique case; evidently in some cases the hisX6 leader simply
folds back onto the desired product so that cutting cannot occur.
3.      You may want to look into the new system that Invitrogen has just
begun to market.  It involves cloning into expression vectors that grow in
the yeast Pichia pastoris.  Two of the vectors provide a leader sequence
that causes the yeast to excrete the product into the media, cleaving off
the leader in the process.  Modifications and folding of the product are
supposed to be properly eukarytic.  According to the references provided by
Invitrogen, at least ten different eukaryotic and prokaryotic proteins have
been produced in this way, and yields average in the GRAMS per liter range.
 If you phone Invitrogen (1-800-95-6288) and ask for Micheal Galleno (X223)
he will FAX you the details.  I may wind up going this route in the long
run even if I do solve my cutting problems with the prokarytic fusion
William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu

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