Hi,
Since you're considering a serious undertaking, it would be good to know
whether your protein is decently expressed and reasonably characterized in
its native state - before advising you on the process :)
If your protein is normally expressed well, is soluble, and not aggregated -
there's going to be little to no difference between the results of major
methods used for primary affinity capture. If your protein is not very
abundant in the lysate, then higher fidelity methods may give you better
purity - in this case StrepII is somewhat better than GST. You can always
try the good old Biotin tag - this will however require that your E. coli
carry excess of BirA (typically supplied from a separate plasmid) as normal
biotinylation levels in most E. coli strains are fairly low.
With 40+ mutants you can expect a certain amount of attrition - some of the
mutants will not express well and some will not purify well.
In general there's nothing wrong with single-step IMAC provided that you can
work out reasonable purification conditions in advance and can assume that
most of your 40+ mutants behave reasonably well (and close to the test
case). For outlier mutants there will undoubtedly be issues with
purification however primary capture method is not likely to fix them since
it did not cause them in the first place.
For abundant proteins - if you use a 'high fidelity' IMAC resin such as
His-SELECT or Talon and if your protein of interest is present (in the
lysate) in reasonable quantity then you can expect the purity of your
single-step product to be compatible between GST and IMAC purification.
StrepII tag is of course a good choice as well.
Regards,
Artem
"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone"
Jorge Luis Borges
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Mark
Collins
Sent: Monday, August 10, 2009 10:37 PM
To: [log in to unmask]
Subject: [ccp4bb] {Spam?} quick purification?
Hi All
A little off topic, but I am thinking about expressing and purifying 40+
mutants, for an assay, at maybe 1mg total protein each. I'd like the
purification to be quick and easy (ie. one step) but cleaner than just a
6his-tag purification.
Currently, my options are either GST, a longer his tag or a strep-tag.
Any thoughts on the comparison between GST and strep and Ni purifications
would be much appreciated. OR are there any other (better) high affinity
purifications, I've missed, excluding expense Ab resins.
Thanks,
Mark Collins
