Mr. M.J. Lush wrote:
Michael,
I can highly recommend the His-tag system for protein expression and
purification. I have heard about some of the problems with the system
but have never been seriously affected by any of them. As far as the
other tags are concerned, the T7 tag is used for little more than
identification later via Western analysis. The S-tag is also used for
this although the Novagen guide indicates that this is also useful in
affinity purification. The Thx-tag (thioredoxin) is used for affinity
purification and there is some conjecture about whether its expression
with another protein, may assist in the correct folding of the alien
protein. I've had no experience with these other systems but have heard
few complaints. The nice thing about having just a his-tag is that you
can usually leave it attached to your protein with little effect on
protein activity and it is unlikely to interfere with structural
studies. If you want to go whole hog though, use the pET32 system which
Novagen has been flogging. It carries the Thx-tag, N-terminal His-tag,
S-tag, MCS, C-terminal His-tag. If you can't purify with this bugger,
then there's something seriously wrong.
As far as your cloning strategy goes, I would be careful about adding
too many extraneous residues to your domain when you engineer it into
the system. You can't really ever be too sure as to what effect these
residues might have on domain activity and/or solubility. We tend to
engineer our domains pretty tightly to the tag or without a tag at all.
As a final thought, check to make sure that your domain does not
contain cleavage sites for whatever protease you are going to use to
remove your tag. Sorry if this sounds like I'm insulting your
intelligence, but you'd be amazed how many people don't think of this
ahead of time.
Good luck,
Randall C Willis, Researcher
Biochemistry, Hosp for Sick Children
3522-555 University Ave.
Toronto, ON
M5G 1X8 CANADA
416-813-5933 (ph) -5022 (fax)
willis at gandalf.psf.sickkids.on.ca
