Comments on Novogen tagging systems
sai at uab.edu
sai at uab.edu
Wed Mar 12 17:54:11 EST 1997
> Would anyone care to comment on the other avalable tags
> (T7.Tag, S.Tag and Trx.tag)?
i have used the pET-32 vector which has the whole shebang i.e all of the
above mentioned tags. it worked great:my typeical yeilds were about
12-13 mg of pure protein per litre of e.coli. since i was going to use
this for crystallography, i should warn you that the thioredoxin
(trx-tag) domain has a very sticky nature in the sense that it tended to
induce oligomerization of my protein. thioredoxin has been implicated
as having chaperone activity and so this was not that surprising in the
sense that it would bind to proteins as a alleged chaperone and thus
induce artifactual oligomerization. i witnessed this with native gels
and during gel filtration runs. also, i really didnt like the choice of
enterokinase as the proteolytic cleaver to obviate the trx-tag.
enterokinase is extremely slow and while it is supposed to be specific,
inevitable trace amounts (sub-picomolar levels) of other contaminating
proteases mainly trypsin in most commercially available enterokinases
will make that cleavage reaction pretty darn messy especially since you
have to incubate the proteolytic reaction for a long time (giving the
femtomolar traces of contaminating proteases plenty of time to wreck
havoc on your protein). so if you have milligram amounts of protein to
cleave, then good luck getting intact protein (i had to incubate my 5 mg
of protein for about 7 days at 25 degrees and i got significant
non-specific and non-intentional cleavage due to contaminating
now, if neither of the above mentioned problmes concern you, i would
highly recommend pET-32, cuz for the amounts of protein you can make
just from one 1 liter prep, it is worth it. also, if you only need
microgram quantities of protein for most of your applications, then you
would probably not need to have long proteolytic times to get rid of the
tag. also, novagen themselves came out with some recombinant
enterokinase kit which is supposed to be goodand specific; i havent
tried it since i've moved on to another system...so if the latter is
your scenario, then go for it. good luck...
Sai (sai at uab.edu) : pre-doctoral fellow i.e lowly graduate student...
Dr.Gerald Hart's lab
Dept. of Biochemistry and Molecular Genetics
University of Alabama at Birmingham...
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