Dear Colleagues,
With regard to the use of GST as a dimerizing fusion partner, the
following paper from my lab provides a well documented example:
https://pubmed.ncbi.nlm.nih.gov/9336840
Cheers, Arne
Am 24.09.20 um 09:27 schrieb Barone, Matthias:
Dear Dhiraj
I just recently had to check how far the proteins lay apart if using
the GST dimerization. We use the pGEX-4T-1 vector and the BamHI
restriction site. If you use a different site, the linker might get a
different length. But to give you a rough feeling, a 110 amino acid
fusion protein (15kDa or so) will be roughly 80A apart. I assume the
might be able to get even closer than that, given the flexibility of
the linker in between (the sequence prior to the Thrombin
cleavage site is DPPKSDLVPR-GS)
Best, Matthias
Dr. Matthias Barone
AG Kuehne, Rational Drug Design
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284
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*From:* CCP4 bulletin board <[email protected]> on behalf of
Schreuder, Herman /DE <[email protected]>
*Sent:* Thursday, September 24, 2020 8:52:14 AM
*To:* [email protected]
*Subject:* [ccp4bb] AW: [ccp4bb] dimeric tag to induce the
homodimerization of protein
Hi Dhiraj,
you could also consider making a fusion protein of your protein with
itself, with a suitable long linker (gly-ser-gly-ser etc.?) in
between. At least that dimer won’t dissociate.
Best,
Herman
*Von:* CCP4 bulletin board <[email protected]> *Im Auftrag von
*Srivastava, Dhiraj
*Gesendet:* Mittwoch, 23. September 2020 21:39
*An:* [email protected]
*Betreff:* [EXTERNAL] Re: [ccp4bb] dimeric tag to induce the
homodimerization of protein
*EXTERNAL : *Real sender is [email protected]
<mailto:[email protected]>
Thanks everyone for all the nice suggestions.
regarding Matthew's question, Yes, There is a possibility of
polydispersity due to the proteins making chain/aggregate, however the
affinity between the dimeric protein A and monomeric protein B is poor
and kinetics of dissociation is very fast thus complex can not survive
gel filtration chromatography. So, by increasing the avidity, We are
hoping that only 2:2 complex will survive the gel filtration and we
will be able to isolate monodispersed complex for further biophysical
studies like SAXS or crystallization.
Thank you
Dhiraj
------------------------------------------------------------------------
*From:*CCP4 bulletin board <[email protected]
<mailto:[email protected]>> on behalf of Gloria Borgstahl
<[email protected] <mailto:[email protected]>>
*Sent:* Tuesday, September 22, 2020 2:28 PM
*To:* [email protected] <mailto:[email protected]>
<[email protected] <mailto:[email protected]>>
*Subject:* Re: [ccp4bb] dimeric tag to induce the homodimerization of
protein
I was thinking the form of GFP that dimerizes. This would also make
it easy to track where the protein is.
On Tue, Sep 22, 2020 at 1:28 PM Diana Tomchick
<[email protected]
<mailto:[email protected]>> wrote:
Any dimeric tag should work if you add a long enough linker to
satisfy your distance criterion.
GST, for example. Download the coordinates and get a rough idea
how long the linker would have to be for your protein.
Diana
**************************************************
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
[email protected]
<mailto:[email protected]>
(214) 645-6383 (phone)
(214) 645-6353 (fax)
On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj
<[email protected] <mailto:[email protected]>>
wrote:
*EXTERNAL MAIL*
Hi
I want to make my protein dimeric to increase its affinity for
its interaction partner which is a dimer. does anyone know a
suitable tag/fusion protein which can be used as C terminal fusion
for this purpose? I can not use any of the leucine zipper as I am
looking for the distance between the c terminus to be around 30-40 A.
Thank you
Dhiraj
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Lehrstuhl f. Biologische Chemie | Technische Universitaet Muenchen
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