pETor pGEX expression => opinions requested.

Randy Willis willis at gandalf.psf.sickkids.on.ca
Tue Mar 11 14:36:36 EST 1997


Chris,

Our lab works with both for different projects and my personal selection
would be to go with the pET system.  There is just something about
adding an entire protein to the end of mine that seems wrong but then,
what do I know.  Both work within limits which are typically set by your
own protein.  The nice thing about a his-tag is that you might be able
to get away with leaving it attached and not have to worry about
introducing a protease to your pure protein.  Also, the his-tag might
help the protein to chelate a heavy metal atom but this I would not be
willing to be quoted on.  I know that Pharmacia has just marketed a new
GST fusion which has a new protease cleavage site which I believe is
supposed to be more specific than thrombin or factor X, but you would
have to check it out...it's called Precission.  Check your protein for
these protease sites as it would be a drag to subclone into a system and
then find that you chop up your protein when you try to remove the
fusion tag.

His-tagged proteins don't come as cleanly from other coli contaminants
on the Ni++ resin although there has been some noise recently about
using Co++ on the column instead and this supposedly gives better
specificity for the tagged protein.  For more on this, check with your
local Clontech people or their web site for something about Talon
Resin.  In any case, I would never rely on just one column and, from
what I can tell, there is less a chance of dimerization with the
his-tags than with the GST fusions.

In any event, it's a crap shoot as both work well.  An idea...if you're
PCRing up the system anyway, why not try both just to hedge your bets?

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



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