Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Michal Taube]

Dear Dhiraj,

You can use coiled-coil domain to dimerize your protein. Please look at those 
exemplary publications:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923517/#R12
https://www.nature.com/articles/nsb877
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375751/
https://pubs.rsc.org/en/content/articlelanding/2017/me/c7me00012j#!divAbstract

Best
Michal
-
Michal Taube, PhD
Department of Macromolecular Physics
Faculty of Physics
A. Mickiewicz University
Uniwersytetu Poznanskiego 2
61-614 Poznan
Poland
mta...@amu.edu.pl



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Prof. Dr. Arne Skerra]

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


*From:* CCP4 bulletin board  on behalf of 
Schreuder, Herman /DE 

*Sent:* Thursday, September 24, 2020 8:52:14 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*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  *Im Auftrag von 
*Srivastava, Dhiraj

*Gesendet:* Mittwoch, 23. September 2020 21:39
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [EXTERNAL] Re: [ccp4bb] dimeric tag to induce the 
homodimerization of protein


*EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk 



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 > on behalf of Gloria Borgstahl 
mailto:gborgst...@gmail.com>>

*Sent:* Tuesday, September 22, 2020 2:28 PM
*To:* CCP4BB@JISCMAIL.AC.UK  
mailto:CCP4BB@JISCMAIL.AC.UK>>
*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 
> 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
diana.tomch...@utsouthwestern.edu

(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj
mailto:dhiraj-srivast...@uiowa.edu>>
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



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



*CAUTION: *This email originated from outside UTSW. Please be
cautious of links or attachments, and validate the sender's email
address before replying.



*UT**Southwestern*

Medical 

[ccp4bb] AW: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Schreuder, Herman /DE]

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  Im Auftrag von Srivastava, 
Dhiraj
Gesendet: Mittwoch, 23. September 2020 21:39
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] dimeric tag to induce the homodimerization of 
protein


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

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 mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Gloria Borgstahl 
mailto:gborgst...@gmail.com>>
Sent: Tuesday, September 22, 2020 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
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 
mailto:diana.tomch...@utsouthwestern.edu>> 
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
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> 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


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
CAUTION: This email originated from outside UTSW. Please be cautious of links 
or attachments, and validate the sender's email address before replying.




UT Southwestern

Medical Center

The future of medicine, today.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Srivastava, Dhiraj]

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  on behalf of Gloria Borgstahl 

Sent: Tuesday, September 22, 2020 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
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 
mailto:diana.tomch...@utsouthwestern.edu>> 
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
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> 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


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

CAUTION: This email originated from outside UTSW. Please be cautious of links 
or attachments, and validate the sender's email address before replying.




UT Southwestern

Medical Center

The future of medicine, today.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Gloria Borgstahl]

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 <
diana.tomch...@utsouthwestern.edu> 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
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj <
> dhiraj-srivast...@uiowa.edu> 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
>
> --
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> CAUTION: This email originated from outside UTSW. Please be cautious of
> links or attachments, and validate the sender's email address before
> replying.
>
>
> --
>
> UT Southwestern
>
> Medical Center
>
> The future of medicine, today.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Tomas Malinauskas]

Hi Dhiraj,

You could try a C-terminal Fc-tag if it is an extracellular protein,
e.g. see pHL-FcHis vector described in the supplement of PMID
17001101.

Best wishes,
Tomas

On Tue, Sep 22, 2020 at 6:08 PM Srivastava, Dhiraj
 wrote:
>
> 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
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Diana Tomchick]

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
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> 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


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

CAUTION: This email originated from outside UTSW. Please be cautious of links 
or attachments, and validate the sender's email address before replying.




UT Southwestern

Medical Center

The future of medicine, today.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] dimeric tag to induce the homodimerization of protein

[Srivastava, Dhiraj]

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



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/