Nicholas is right that it depends a lot on the dimer you are working
with. I worked on a dimeric tRNA synthetase and was able to make
heterodimers in a couple of ways. The most effective was to make a
bicistronic version of the ORFs each with their own S-D. Each had a tag
for tandem affinity purification as Nick suggests. In my case, using two
plasmids or even putting the two ORFs with separate promoters did not
result in heterodimers (probably due to folding/dimerization during
translation?). I was able to express to the two dimeric versions
separately, mix, dilute extensively (which ultimately resulted in a big
hit in yield), concentrate, and purify through the tandem approach. I
eventually noticed that even though it was slow, there was dimer
exchange occurring at intermediate concentrations. I ended up
engineering a disulfide at the dimer interface to minimize that.
--paul
On 08/27/2013 01:10 PM, Noinaj, Nicholas (NIH/NIDDK) [F] wrote:
Anindito,
First off, I have never tried this, but here are a few initial thoughts on how
I might approach this. But first a question, how tight is the dimer form? if
you mixed dimers with tags and dimers without tags, would you get any exchange?
If so, you might be able to use this to your advantage. But for now, i'll
ignore this.
Now for how i was thinking of possibly accomplishing the goal you want...
1- use two vectors or a DUET vector to express two versions of your protein,
(1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2)
with N-term cleavable (TEV site?) GST tag only.
2- express your protein as normal and then purify by (1) running the GSH column
to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture
those with HIS tags. If all works as planned, you would end up with one
monomer having only HIS-fusion protein tags and one having GST tag. You could
use SDS-PAGE/Western blot analysis to verify this, since the monomers will run
at different sizes (you might have to modify your gel system to get the best
separation....i recommend 5-7% gels for this size in MES) AND should be
reactive to antiHIS and antiGST antibodies in needed. Further, you could get
an idea for the ratios of each by comparing the overall intensity of the bands
under normal staining methods.
3- to remove the HIS and GST tags, just treat with TEV protease to yield your
dimer with only one of the monomers containing your fusion protein.
I am sure i likely overlooked something in my haste, but you get the idea,
basically a two step purification with two tags. Again, I haven't tried this
but seems like it should work to me. hope this helps, good luck!
Cheers,
Nick
--------------------------------
[ Nicholas Noinaj ]
the Buchanan Lab
Laboratory of Molecular Biology
LMB-NIDDK, NIH
50 South Drive, Room 4505
Bethesda, MD 20892-8030
1-301-594-9230 (lab)
1-859-893-4789 (cell)
[email protected]
[ the Buchanan Lab ]
http://www-mslmb.niddk.nih.gov/buchanan/
________________________________________
From: Anindito Sen [[email protected]]
Sent: Monday, August 26, 2013 11:26 PM
To: [email protected]
Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer
protein complex
Dear All,
I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer
protein (size is ~100kDa). To be more precise - The tag need to get itself
attached only on one of the dimer molecules.
My expertise are not in Cell biology and therefore any suggestion in this
regard will be of great help.
Thanks & Best Wishes
Dr. Anindito Sen (Ph.D)
Structural Biology
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo. Bunkyo-ku. Tokyo
113-0033. Japan
Tel & Fax: +81-3-5841-3339
