Re: [ccp4bb] For stabilizing protein-protein complex

[Debanu]

Hi Sanjeev,

In addition to all the excellent suggestions (trying different buffers/salts, 
co-expression, concentrator, incubate and set up crystallization screening, 
checking SPR, fusion), I have a couple more suggestions:

1) related to your question about cross-linker: do you have experimental 
structures or homology models of one or both components? If so, you could try a 
couple of protein-protein docking programs (Haddock, etc) and analyze the 
interface for close contacts where you can introduce disulfides.

2) you can also try lysing the cells together and co-purifying from the 
beginning.

3) try reductive methylation on the incubated complex prior to setting up 
screens if you have numerous exposed lysines.

Thanks,
Debanu
Accelero Biostructures 

> On Jan 31, 2017, at 8:05 AM, sanjeev kumar  wrote:
> 
> Dear all,
> 
> I am trying to stabilize a protein-protein complex. Our SPR study indicates 
> it is having micro molar dissociation constant. I tried to purify both the 
> molecule in complex form with size exclusion chromatography (mixed both the 
> protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt 
> observed formation of complex as both the molecule eluted at their respected 
> elution volume.
> Please suggest me to get a better way to achieve the complex and if anyone 
> gives idea about what is the good cross-linker I can use.
> Suggestions are highly appreciated.
> 
> Thanks
> 
> best
> sanjeev kumar, PhD
> Purdue University
> West Lafayette
> Indiana
> 
> 

Re: [ccp4bb] For stabilizing protein-protein complex

[Radisky, Evette S., Ph.D.]

I agree with Engin’s suggestions.

Our group crystallized a complex of mesotypsin with BPTI, where we measured an 
inhibition constant (approximating Kd) of 14 micromolar, by mixing the two pure 
proteins together in 1:1 stoichiometry.  Actually we do that for a lot of our 
complexes with higher affinity also.  It is definitely worth a try.

http://www.jbc.org/content/283/7/4115.full

Evette

Evette S. Radisky, Ph.D.
Associate Professor and Consultant
Department of Cancer Biology
Mayo Clinic Cancer Center
_
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372
http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Engin 
Özkan
Sent: Tuesday, January 31, 2017 1:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] For stabilizing protein-protein complex


Not observing a complex on a gel filtration run for a week complex (micromolar) 
is not necessarily unexpected. It all depends on your protein concentrations, 
the dissociation kinetics (remember, a gel filtration run is not an equilibrium 
experiment - it dilutes and separates your sample), etc.

I would make sure that the SPR results are believable and sensible: if the 
affinity is really weak, I'd expect very fast kinetics - most weak interactions 
come with faster association and dissociation. As a result, in this affinity 
range, you can't fit kinetics data in SPR, and can only do an Langmuir isotherm 
fit to your equilibrium binding response values.

Also, you should check your expected Rmax values (based on how much you 
captured on the chip), and make sure that your responses don't exceed the 
theoretical values. Otherwise, you are just observing non-specific binding. 
Similarly, if your responses are way too low compared to the expected, your 
protein on the chip might be mostly inactive - again, not good.

If everything checks out, just mix your two proteins at the right 
stoichiometric ratio, concentrate a lot, and go ahead with crystallization (if 
that's what you want).

Engin

On 1/31/17 10:05 AM, sanjeev kumar wrote:
Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study indicates it 
is having micro molar dissociation constant. I tried to purify both the 
molecule in complex form with size exclusion chromatography (mixed both the 
protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt 
observed formation of complex as both the molecule eluted at their respected 
elution volume.
Please suggest me to get a better way to achieve the complex and if anyone 
gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana

Re: [ccp4bb] For stabilizing protein-protein complex

[Engin Özkan]

Not observing a complex on a gel filtration run for a week complex 
(micromolar) is not necessarily unexpected. It all depends on your 
protein concentrations, the dissociation kinetics (remember, a gel 
filtration run is not an equilibrium experiment - it dilutes and 
separates your sample), etc.


I would make sure that the SPR results are believable and sensible: if 
the affinity is really weak, I'd expect very fast kinetics - most weak 
interactions come with faster association and dissociation. As a result, 
in this affinity range, you can't fit kinetics data in SPR, and can only 
do an Langmuir isotherm fit to your equilibrium binding response values.


Also, you should check your expected Rmax values (based on how much you 
captured on the chip), and make sure that your responses don't exceed 
the theoretical values. Otherwise, you are just observing non-specific 
binding. Similarly, if your responses are way too low compared to the 
expected, your protein on the chip might be mostly inactive - again, not 
good.


If everything checks out, just mix your two proteins at the right 
stoichiometric ratio, concentrate a lot, and go ahead with 
crystallization (if that's what you want).


Engin


On 1/31/17 10:05 AM, sanjeev kumar wrote:

Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study 
indicates it is having micro molar dissociation constant. I tried to 
purify both the molecule in complex form with size exclusion 
chromatography (mixed both the protein in equal molar ratio and 
incubated at 4 degree for 1 hour), I didnt observed formation of 
complex as both the molecule eluted at their respected elution volume.
Please suggest me to get a better way to achieve the complex and if 
anyone gives idea about what is the good cross-linker I can use.

Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana

Re: [ccp4bb] For stabilizing protein-protein complex

[Tanner, John J.]

If your goal is a crystal structure, this reference may be worth consulting.  
The paper describes the use of centrifugal concentrators to make a complex of 
two proteins that have Kd of 19 micromolar for crystallization.  They mixed the 
two proteins and then concentrated using a membrane that allows passage of the 
smaller protein while retaining the complex.  The protein-protein complex 
crystallized.

Ignatev A, Piatkov K, Pylypenko O, Rak A. A size filtration approach to purify
low affinity complexes for crystallization. J Struct Biol. 2007 
Jul;159(1):154-7.
PubMed PMID: 17408969.


John J. Tanner
Professor of Biochemistry and Chemistry
Chair, Biochemistry Department Graduate Admissions Committee
Department of Biochemistry
University of Missouri-Columbia
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Fax: 573-882-5635
Email: tanne...@missouri.edu
http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A

On Jan 31, 2017, at 10:05 AM, sanjeev kumar 
>
 wrote:

Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study indicates it 
is having micro molar dissociation constant. I tried to purify both the 
molecule in complex form with size exclusion chromatography (mixed both the 
protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt 
observed formation of complex as both the molecule eluted at their respected 
elution volume.
Please suggest me to get a better way to achieve the complex and if anyone 
gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana

Re: [ccp4bb] For stabilizing protein-protein complex

[Debasish Kumar Ghosh]

Dear Sanjeev,

You can try to clone both of your gene in pET duet vector to coexpress both 
proteins in bacterial expression system and purify with Nickel affinity 
exchange chromatography (both proteins will be his tagged). This will eliminate 
the problem of critical mixing of both proteins in stochiometric ratio as in 
vivo condition (i.e. bacterial cell) will take care of that. 
Other wise, you may determine the stochiometric binding ratio from SPR data 
(like by using Langmuir or other models) and mix the individual proteins 
accordingly. 
Buffer (and ions) play some crucial role in some protein protein interaction. 
So you may also try to play around with different buffer and salt compositions 
(if you intuitively guess the ion or buffer conditions).
As the Kd value is in micro molar range, it seems it is not of very strong 
affinity kind of interaction. So longer incubation (4 degree/ rotation) might 
give at least formation of some complex. 
Hope this helps. 

Best!!



Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: sanjeev kumar 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tue, 31 Jan 2017 21:35:32 +0530 (IST)
Subject: [ccp4bb] For stabilizing protein-protein complex

Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study indicates
it is having micro molar dissociation constant. I tried to purify both the
molecule in complex form with size exclusion chromatography (mixed both the
protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt
observed formation of complex as both the molecule eluted at their
respected elution volume.
Please suggest me to get a better way to achieve the complex and if anyone
gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana

Re: [ccp4bb] For stabilizing protein-protein complex

[Napoleao Fonseca Valadares]

If your SPR data is correct, you'll need to use a micro molar protein 
concentration (or higher due to dilution in the column) in your analytical size 
exclusion chromatography assay. What concentration did you use? Also, ideally 
the buffers in both assays should match. And maybe your SPR data is the source 
of confusion (it is not uncommon). What is the shape of your sensorgrams? Do 
you measure the dissociation for how long, or does it happens abruptly? 
Napo 

- Mensagem original -

> De: "sanjeev kumar" 
> Para: CCP4BB@JISCMAIL.AC.UK
> Enviadas: Terça-feira, 31 de Janeiro de 2017 14:05:32
> Assunto: [ccp4bb] For stabilizing protein-protein complex

> Dear all,

> I am trying to stabilize a protein-protein complex. Our SPR study
> indicates it is having micro molar dissociation constant. I tried to
> purify both the molecule in complex form with size exclusion
> chromatography (mixed both the protein in equal molar ratio and
> incubated at 4 degree for 1 hour), I didnt observed formation of
> complex as both the molecule eluted at their respected elution
> volume.
> Please suggest me to get a better way to achieve the complex and if
> anyone gives idea about what is the good cross-linker I can use.
> Suggestions are highly appreciated.

> Thanks

> best
> sanjeev kumar, PhD
> Purdue University
> West Lafayette
> Indiana

Re: [ccp4bb] For stabilizing protein-protein complex

[Keller, Jacob]

You could also try co-expressing them, pull down the complex by his tag. Also 
you can try equilibrating the SEC column in a low concetration of one of the 
proteins, if you can express enough. It is a bit surprising that you saw no 
peak for the complex though. Since you have SPR data, what are kon and 
koff—fast I assume?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ursula 
Schulze-Gahmen
Sent: Tuesday, January 31, 2017 11:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] For stabilizing protein-protein complex

We have had good luck with creating fusion proteins of the 2 proteins in 
question (http://www.nature.com/articles/ncomms14076). If you don't know how 
they interact, you would need to try different linker length and different 
order of the 2 proteins in the fusion protein. It would also be helpful to have 
a way to assay for the functional complex.
Ursula Schulze-Gahmen

On Tue, Jan 31, 2017 at 8:05 AM, sanjeev kumar 
<sanjeev@gmail.com<mailto:sanjeev@gmail.com>> wrote:
Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study indicates it 
is having micro molar dissociation constant. I tried to purify both the 
molecule in complex form with size exclusion chromatography (mixed both the 
protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt 
observed formation of complex as both the molecule eluted at their respected 
elution volume.
Please suggest me to get a better way to achieve the complex and if anyone 
gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana





--
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

Re: [ccp4bb] For stabilizing protein-protein complex

[Ursula Schulze-Gahmen]

We have had good luck with creating fusion proteins of the 2 proteins in
question (http://www.nature.com/articles/ncomms14076). If you don't know
how they interact, you would need to try different linker length and
different order of the 2 proteins in the fusion protein. It would also be
helpful to have a way to assay for the functional complex.

Ursula Schulze-Gahmen

On Tue, Jan 31, 2017 at 8:05 AM, sanjeev kumar 
wrote:

> Dear all,
>
> I am trying to stabilize a protein-protein complex. Our SPR study
> indicates it is having micro molar dissociation constant. I tried to purify
> both the molecule in complex form with size exclusion chromatography (mixed
> both the protein in equal molar ratio and incubated at 4 degree for 1
> hour), I didnt observed formation of complex as both the molecule eluted at
> their respected elution volume.
> Please suggest me to get a better way to achieve the complex and if anyone
> gives idea about what is the good cross-linker I can use.
> Suggestions are highly appreciated.
>
> Thanks
>
> best
> sanjeev kumar, PhD
> Purdue University
> West Lafayette
> Indiana
>
>
>


-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491