Re: [ccp4bb] crystallizing fusion proteins

[Bostjan Kobe]

Hi Herman

I agree with all discussed already.

First, is your protein of interest actually folded or just solubilized by the 
large fusion partner?

If it is folded, worth considering crystallization of the fusion protein, as a 
“desperate measure” approach. The key issue will be making the linkage between 
the proteins reasonably rigid, to increase chances of crystallization. In 
GPCRs, T4L is usually inserted into a loop, which obviously will constrain it 
more than just one linkage at the terminus.

There is quite a bit of literature on the topic already, and some nice tools 
like the MBP with increased crystallization propensity, as already pointed out. 
Let me know if I can help finding any key papers.

But at the end of the day, my feeling is people try this a lot, as setting up 
some crystallization plates is easier than troubleshooting cleavage, 
purification and making and purifying new constructs. Despite this, other than 
specific cases like GPCRs, there are not that many successful examples really. 
So my feeling is this approach doesn’t work that often without a lot of trial 
and optimization.

Best wishes

Bostjan

--
Bostjan Kobe FAA
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Professor of Structural Biology
School of Chemistry and Molecular Biosciences
and Institute for Molecular Bioscience (Division of Chemistry and Structural 
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From: CCP4 bulletin board  on behalf of Tao-Hsin Chang 

Reply to: Tao-Hsin Chang 
Date: Tuesday, 16 March 2021 at 6:55 am
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] crystallizing fusion proteins

Hi Herman,

We learned a few tricks of using MBP fusion for solving an interesting fold of 
a receptor extracellular domain from our recent publication 
(https://pubmed.ncbi.nlm.nih.gov/32541044/).
(1) the length of a linker between MBP and protein of interest is critical. Use 
a computational modeling approach to figure out a reasonable linker, if 
possible.
(2) take advantage of engineered MBP e.g., surface entropy reduction mutations 
and ion-mediated dimerization mutations (a good review 
https://pubmed.ncbi.nlm.nih.gov/26682969/; 
https://pubmed.ncbi.nlm.nih.gov/26850170/) that do help.
(3) use either E. coli Shuffle cells or mammalian cells to deal with the 
disulfide bonds and characterize the protein folding e.g. CD or SEC (not ideal, 
but simple).

Hope this helps.

Best wishes,
Tao-Hsin

Tao-Hsin Chang, DPhil
Research Specialist
Howard Hughes Medical Institute



On Mar 15, 2021, at 5:07 AM, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear Bulletin Board,
Sorry for the slightly off-topic question, but we are struggling with a 
receptor domain that expresses well as a fusion protein, but gets lost the 
moment it is cleaved from the fusion partner. It could be that the receptor 
domain is not or misfolded, but it could also be a solubility problem.

I have seen some crystal structures of fusion proteins with MBP and for 
membrane proteins, T4-lysozyme fusions are often crystallized. What is your 
experience? Would it be worth trying to express and crystallize a fusion 
protein, or would it be better to look for other constructs, e.g. to include 
more receptor domains?

Thank you very much for your advice!
Herman





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Re: [ccp4bb] crystallizing fusion proteins

[Tao-Hsin Chang]

Hi Herman,

We learned a few tricks of using MBP fusion for solving an interesting fold of 
a receptor extracellular domain from our recent publication 
(https://pubmed.ncbi.nlm.nih.gov/32541044/ 
).
(1) the length of a linker between MBP and protein of interest is critical. Use 
a computational modeling approach to figure out a reasonable linker, if 
possible.
(2) take advantage of engineered MBP e.g., surface entropy reduction mutations 
and ion-mediated dimerization mutations (a good review 
https://pubmed.ncbi.nlm.nih.gov/26682969/ 
; 
https://pubmed.ncbi.nlm.nih.gov/26850170/ 
) that do help.
(3) use either E. coli Shuffle cells or mammalian cells to deal with the 
disulfide bonds and characterize the protein folding e.g. CD or SEC (not ideal, 
but simple).

Hope this helps.

Best wishes,
Tao-Hsin

Tao-Hsin Chang, DPhil
Research Specialist
Howard Hughes Medical Institute
 

> On Mar 15, 2021, at 5:07 AM, Schreuder, Herman /DE 
>  wrote:
> 
> Dear Bulletin Board,
> Sorry for the slightly off-topic question, but we are struggling with a 
> receptor domain that expresses well as a fusion protein, but gets lost the 
> moment it is cleaved from the fusion partner. It could be that the receptor 
> domain is not or misfolded, but it could also be a solubility problem.
>  
> I have seen some crystal structures of fusion proteins with MBP and for 
> membrane proteins, T4-lysozyme fusions are often crystallized. What is your 
> experience? Would it be worth trying to express and crystallize a fusion 
> protein, or would it be better to look for other constructs, e.g. to include 
> more receptor domains?  
>  
> Thank you very much for your advice!
> Herman
>  
>  
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] [EXT] Re: [ccp4bb] crystallizing fusion proteins

[Syed,Aleem]

As others suggested, I would check the globularity of the fusion protein with 
biophysical technique such as SAXS, if possible.

Aleem

On Mar 15, 2021, at 10:52 AM, Pascal Egea 
<4aa44fc90f38-dmarc-requ...@jiscmail.ac.uk> wrote:


WARNING: This email originated from outside of MD Anderson. Please validate the 
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Hi Herman,
I will add a few points to all the excellent advice already provided to you.
If you decide to try this option of crystallizing a fusion protein of your 
target, I would consider using N-terminal but also C-terminal fusions. We have 
had success using MBP in N-ter and the superfolder GFP in C-terminal for a few 
'vexing' proteins from Plasmodium falciparum. It helped us either to obtain 
crystals/structures (as you seek) but also provided a way to circumvent a 
tendency for twinning from our target in some specific cases.
GFP has the added benefit that your fusion crystals should be bright yellow so 
that speeds up the screening process a bit.

I hope this helps. Good luck
Best regards,

Pascal Egea, PhD
UCLA School of Medicine






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Re: [ccp4bb] crystallizing fusion proteins

[Michael Hothorn]

For us, a C-terminal macro domain tag worked well to crystallize a 
rather flexible target protein.


https://pubmed.ncbi.nlm.nih.gov/27774698/

best regards,

Michael

On 15.03.21 16:51, Pascal Egea wrote:

Hi Herman,
I will add a few points to all the excellent advice already provided 
to you.
If you decide to try this option of crystallizing a fusion protein of 
your target, I would consider using N-terminal but also C-terminal 
fusions. We have had success using MBP in N-ter and the superfolder 
GFP in C-terminal for a few 'vexing' proteins from /Plasmodium 
falciparum. /It helped us either to obtain crystals/structures (as you 
seek) but also provided a way to circumvent a tendency for twinning 
from our target in some specific cases.
GFP has the added benefit that your fusion crystals should be 
bright yellow so that speeds up the screening process a bit.


I hope this helps. Good luck
Best regards,

Pascal Egea, PhD
UCLA School of Medicine





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Re: [ccp4bb] crystallizing fusion proteins

[Pascal Egea]

Hi Herman,
I will add a few points to all the excellent advice already provided to you.
If you decide to try this option of crystallizing a fusion protein of your
target, I would consider using N-terminal but also C-terminal fusions. We
have had success using MBP in N-ter and the superfolder GFP in C-terminal
for a few 'vexing' proteins from *Plasmodium falciparum. *It helped us
either to obtain crystals/structures (as you seek) but also provided a way
to circumvent a tendency for twinning from our target in some specific
cases.
GFP has the added benefit that your fusion crystals should be bright yellow
so that speeds up the screening process a bit.

I hope this helps. Good luck
Best regards,

Pascal Egea, PhD
UCLA School of Medicine


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






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Re: [ccp4bb] crystallizing fusion proteins

[Peat, Tom (Manufacturing, Parkville)]

Hello Herman,

If you have plenty of the fusion protein, there is probably no reason not to 
set it up in crystallisation trials and see if you get something.
There are published reports of proteins crystallising with fusion partners, but 
I suspect there are a whole lot of unpublished results of this not working as 
well.
As Artem has already stated, it could be that your protein of interest just 
doesn't like the buffer conditions when it finds itself alone in solution after 
cleavage, so that is an area to explore.
Best of luck, tom

Tom Peat, PhD
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Schreuder, 
Herman /DE 
Sent: Monday, March 15, 2021 8:07 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] crystallizing fusion proteins


Dear Bulletin Board,

Sorry for the slightly off-topic question, but we are struggling with a 
receptor domain that expresses well as a fusion protein, but gets lost the 
moment it is cleaved from the fusion partner. It could be that the receptor 
domain is not or misfolded, but it could also be a solubility problem.



I have seen some crystal structures of fusion proteins with MBP and for 
membrane proteins, T4-lysozyme fusions are often crystallized. What is your 
experience? Would it be worth trying to express and crystallize a fusion 
protein, or would it be better to look for other constructs, e.g. to include 
more receptor domains?



Thank you very much for your advice!

Herman









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Re: [ccp4bb] crystallizing fusion proteins

[Artem Evdokimov]

If you have a biophysical way to confirm functionality and/or folding it
would go a long way towards helping you make this choice.

Does your domain bind a ligand of any kind? Is it significantly stable in a
simple thermal melt experiment (of course you have to account for the
melting of the fusion partner)? What are the symptoms of precipitation or
"disappearance" upon cleavage? How far did you explore the buffer
composition for the cleaved material?

I have seen a whole spectrum of situations like yours from a nasty
misfolded protein kept afloat by fusion, to a perfectly folded domain that
simply did not like the buffer conditions and was essentially insoluble
after cleavage due to its bull properties' mismatch with those of the
solvent.

Artem

On Mon, Mar 15, 2021, 5:08 AM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Dear Bulletin Board,
>
> Sorry for the slightly off-topic question, but we are struggling with a
> receptor domain that expresses well as a fusion protein, but gets lost the
> moment it is cleaved from the fusion partner. It could be that the receptor
> domain is not or misfolded, but it could also be a solubility problem.
>
>
>
> I have seen some crystal structures of fusion proteins with MBP and for
> membrane proteins, T4-lysozyme fusions are often crystallized. What is your
> experience? Would it be worth trying to express and crystallize a fusion
> protein, or would it be better to look for other constructs, e.g. to
> include more receptor domains?
>
>
>
> Thank you very much for your advice!
>
> Herman
>
>
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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[ccp4bb] crystallizing fusion proteins

[Schreuder, Herman /DE]

Dear Bulletin Board,
Sorry for the slightly off-topic question, but we are struggling with a 
receptor domain that expresses well as a fusion protein, but gets lost the 
moment it is cleaved from the fusion partner. It could be that the receptor 
domain is not or misfolded, but it could also be a solubility problem.

I have seen some crystal structures of fusion proteins with MBP and for 
membrane proteins, T4-lysozyme fusions are often crystallized. What is your 
experience? Would it be worth trying to express and crystallize a fusion 
protein, or would it be better to look for other constructs, e.g. to include 
more receptor domains?

Thank you very much for your advice!
Herman






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[ccp4bb] Research Fellow position at A*STAR in Singapore

[hans song]

*Research Fellow Position in Structural Biology*



*Date available: immediately*



*Date posted: 15 March 2021*



We are seeking an enthusiastic and self-motivated postdoctoral research
fellow to work in laboratory of structural  biology for signalling and
diseases in Institute of Molecular and Cell Biology, A*STAR, Singapore. We
take a combinatorial approach of molecular biology, biochemical and
biophysical methods, and structural biology (crystallography and cryo-EM)
to study proteins in the Hippo signaling pathway.Singapore is an excellent
environment to do research with many research groups close by in Biopolis [
https://www.a-star.edu.sg/imcb]. A*STAR offers highly competitive salaries
with benefits including housing allowance and 1-3 months performance bonus.



Requirements:

·   PhD in biochemistry, biophysics, molecular and cell biology or  a
related field

·   Experience in Cryo-EM and/or X-ray crystallography or in molecular
docking, compound design and molecular dynamics simulation

·  Practical skills in molecular biology, protein expression
(preferably insect and/or mammalian cells)and purification, and various
biochemical techniques

·  Good publication record

·  Competent in scientific writing



Interested applicants should send a CV, publication list, a brief
description of research interests and experience, and three confidential
letters of reference to: Prof. Haiwei Song (Email: hai...@imcb.a-star.edu.sg
),
Institute of Molecular and Cell Biology, A*STAR, Singapore.



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