Re: [ccp4bb] TEV vs HRV3C

[Artem Evdokimov]

One final comment: if you need orthogonal proteases, TEV and TVMV do not
seem to cut each others' sites (at least for me they don't). No idea if 3C
will cut both, or only TEV site.

Artem

- Cosmic Cats approve of this message


On Thu, Dec 8, 2022 at 5:26 AM Dom Bellini - MRC LMB <
dbell...@mrc-lmb.cam.ac.uk> wrote:

> Hi All,
>
> I agree with everything said and I also find 3C, my first choice, to
> cleave more efficiently/rapidly. But I remember all the folks working with
> membrane proteins were using TEV, so I wonder whether TEV may be more
> resistent to detergent? (but I never read/found any info about this).
>
> BW,
>
> D
>
>
>
> On 08/12/2022 05:40, Nikolay Dobrev wrote:
>
> Dear all,
> I agree with all said so far:
> Here are some points from my experience and discussion on this topic.
>
> Most of the lab's due use TEV or 3C, depending on the cleavage site in
> their constructs and make their purification strategy work with what they
> have.
>
> Here are some points from my side:
>
>
>- 3C protease is much faster in cleaving the fusion protein in the
>1:100 mass ratio (a test we use in cleavage protocols, even though we
>should use a molar ratio. Consider the difference between 20 kDa protein
>and 200 kDa protein, it is 10x in the amount of substrate difference) 3C
>cleaves in 30 mins more than 95% of the protein at 4-8C. TEV does need much
>longer, and as default, people go for overnight dialysis and cleavage
>simultaneously. Sometimes that can be too long for sensitive proteins and
>lead to precipitation due to chance in the buffer salt concentration etc,
>for an extended period. Quite prominently is observed for Nucleic acid
>binding protein, due to lowering the salt for IEX column and precipitation
>surprise overnight.
>- Neither 3C nor TEV requires a reducing agent, which is essential to
>know as many people are working with S-S-containing proteins, and they take
>an aliquot of the protease purified in the lab without knowing there was a
>reducing agent in the storage buffer. Then surprises can be observed.
>Consider the concentration of 1 mM DTT; even 10 or 100 diluted is 10 µM at
>least.
>- Same with a chelating agent like EDTA, no need to be added to the
>storage buffer; the danger comes when you cleave ion-containing proteins
>like Zn-fingers.
>- 3C is much more efficient in on/column cleavage (as was already
>mentioned). This simple trick gives much better purity than Imidazole
>elution, especially when the Ni-column is not saturated and many
>contaminants are bound. Releasing the protein with the protease makes it
>much cleaner. Most of the protease has a His-tag, which allows at the same
>time to bind it to do beads (of course this slows down a bit the cleavage
>therefore mixing is required). Alternatively, GST-(only)-tag protease is
>better used for the cleavage on Ni beads and then captured on the GSH 
> resin.
>- 3C protease is much more efficient for cleaving in the presence of
>detergent, there is an extensive study on that topic (
>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/)
>- Both proteases are easy to produce, as mentioned, and easily 100-300
>mg can be obtained from 1 L culture (TB or auto-induction with OD= 8-16)
>- 3C protease is not happy at high protein concentrations (>4 mg/ml)
>in low salt buffer (150 mM). Therefore huge loss is observed if, during the
>Ni-elution, low salt buffer is used as the protein comes from the Ni-NTA at
>a concentration 15-20 mg/ml. Please use at least 500 mM NaCl for that step
>or better for any step from lysis to elution.
>- And yes both proteases have cross-activity and can cleave the
>opposite target site with lower activity.
>
> These are the few points coming from the top of my head.
> I hope they are helpful and wish everyone happy cleaving of the fusion
> proteins :)
>
> Kind regards,
> Nikolay
>
> *Nikolay Dobrev *
> Postdoctoral Fellow @ Wilmanns group
> EMBL Hamburg, c/o DESY, Building 25A,
> Notkestraße 85, 22607 Hamburg, Germany
> T +49 40 89902 165 | M +49 173 684 0532
> twitter.com/emblevents | facebook.com/embl.org |
> youtube.com/user/emblmedia
> Visit www.embl.org/events for a complete list of all EMBL events.
>
> On 12/07/2022 11:51 PM Lior Almagor 
>  wrote:
>
>
> In my experience, 3C can partially cut TEV sites as well. If using
> sequentially, it is best to plan to use the TEV first.
>
> On Dec 7, 2022, at 2:42 PM, Lau Kelvin <
> 5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk> wrote:
> I agree. I think it depends on the lab and vectors and personal
> preference.
>
> But David, have you used them sequentially? I have once tried to make a
> His-GST-ENLYFQ-3C construct, and I found that it was self cleaving during
> expression. However I have never tried to replicate the results in vitro.
>
>
>
> --
> Kelvin Lau
> Protein production and structure core 

Re: [ccp4bb] TEV vs HRV3C

[Dom Bellini - MRC LMB]

Hi All,

I agree with everything said and I also find 3C, my first choice, to 
cleave more efficiently/rapidly. But I remember all the folks working 
with membrane proteins were using TEV, so I wonder whether TEV may be 
more resistent to detergent? (but I never read/found any info about this).


BW,

D



On 08/12/2022 05:40, Nikolay Dobrev wrote:

Dear all,
I agree with all said so far:
Here are some points from my experience and discussion on this topic.

Most of the lab's due use TEV or 3C, depending on the cleavage site in 
their constructs and make their purification strategy work with what 
they have.


Here are some points from my side:

  * 3C protease is much faster in cleaving the fusion protein in the
1:100 mass ratio (a test we use in cleavage protocols, even though
we should use a molar ratio. Consider the difference between 20
kDa protein and 200 kDa protein, it is 10x in the amount of
substrate difference) 3C cleaves in 30 mins more than 95% of the
protein at 4-8C. TEV does need much longer, and as default, people
go for overnight dialysis and cleavage simultaneously. Sometimes
that can be too long for sensitive proteins and lead to
precipitation due to chance in the buffer salt concentration etc,
for an extended period. Quite prominently is observed for Nucleic
acid binding protein, due to lowering the salt for IEX column and
precipitation surprise overnight.
  * Neither 3C nor TEV requires a reducing agent, which is essential
to know as many people are working with S-S-containing proteins,
and they take an aliquot of the protease purified in the lab
without knowing there was a reducing agent in the storage buffer.
Then surprises can be observed. Consider the concentration of 1 mM
DTT; even 10 or 100 diluted is 10 µM at least.
  * Same with a chelating agent like EDTA, no need to be added to the
storage buffer; the danger comes when you cleave ion-containing
proteins like Zn-fingers.
  * 3C is much more efficient in on/column cleavage (as was already
mentioned). This simple trick gives much better purity than
Imidazole elution, especially when the Ni-column is not saturated
and many contaminants are bound. Releasing the protein with the
protease makes it much cleaner. Most of the protease has a
His-tag, which allows at the same time to bind it to do beads (of
course this slows down a bit the cleavage therefore mixing is
required). Alternatively, GST-(only)-tag protease is better used
for the cleavage on Ni beads and then captured on the GSH resin.
  * 3C protease is much more efficient for cleaving in the presence of
detergent, there is an extensive study on that topic
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/)
  * Both proteases are easy to produce, as mentioned, and easily
100-300 mg can be obtained from 1 L culture (TB or auto-induction
with OD= 8-16)
  * 3C protease is not happy at high protein concentrations (>4 mg/ml)
in low salt buffer (150 mM). Therefore huge loss is observed if,
during the Ni-elution, low salt buffer is used as the protein
comes from the Ni-NTA at a concentration 15-20 mg/ml. Please use
at least 500 mM NaCl for that step or better for any step from
lysis to elution.
  * And yes both proteases have cross-activity and can cleave the
opposite target site with lower activity.

These are the few points coming from the top of my head.
I hope they are helpful and wish everyone happy cleaving of the fusion 
proteins :)


Kind regards,
Nikolay

*Nikolay Dobrev *
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents  | 
facebook.com/embl.org  | 
youtube.com/user/emblmedia 
Visit www.embl.org/events  for a complete 
list of all EMBL events.

On 12/07/2022 11:51 PM Lior Almagor  wrote:


In my experience, 3C can partially cut TEV sites as well. If using 
sequentially, it is best to plan to use the TEV first.


On Dec 7, 2022, at 2:42 PM, Lau Kelvin 
<5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk> wrote:
I agree. I think it depends on the lab and vectors and personal 
preference.


But David, have you used them sequentially? I have once tried to 
make a His-GST-ENLYFQ-3C construct, and I found that it was self 
cleaving during expression. However I have never tried to replicate 
the results in vitro.




--
Kelvin Lau
Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch 
Phone: +41 21 69 34494

On 7 Dec 2022, at 21:38, David Briggs  
wrote:

Hi Gloria,

Both can be made very easily in E.coli.
Both are active at 4°C, but especially 3C, I think.

Re: [ccp4bb] TEV vs HRV3C

[Nikolay Dobrev]

Dear all,
I agree with all said so far:
Here are some points from my experience and discussion on this topic.

Most of the lab's due use TEV or 3C, depending on the cleavage site in their 
constructs and make their purification strategy work with what they have.

Here are some points from my side:

* 3C protease is much faster in cleaving the fusion protein in the 1:100 mass 
ratio (a test we use in cleavage protocols, even though we should use a molar 
ratio. Consider the difference between 20 kDa protein and 200 kDa protein, it 
is 10x in the amount of substrate difference) 3C cleaves in 30 mins more than 
95% of the protein at 4-8C. TEV does need much longer, and as default, people 
go for overnight dialysis and cleavage simultaneously. Sometimes that can be 
too long for sensitive proteins and lead to precipitation due to chance in the 
buffer salt concentration etc, for an extended period. Quite prominently is 
observed for Nucleic acid binding protein, due to lowering the salt for IEX 
column and precipitation surprise overnight.
* Neither 3C nor TEV requires a reducing agent, which is essential to know as 
many people are working with S-S-containing proteins, and they take an aliquot 
of the protease purified in the lab without knowing there was a reducing agent 
in the storage buffer. Then surprises can be observed. Consider the 
concentration of 1 mM DTT; even 10 or 100 diluted is 10 µM at least. 
* Same with a chelating agent like EDTA, no need to be added to the storage 
buffer; the danger comes when you cleave ion-containing proteins like 
Zn-fingers.
* 3C is much more efficient in on/column cleavage (as was already mentioned). 
This simple trick gives much better purity than Imidazole elution, especially 
when the Ni-column is not saturated and many contaminants are bound. Releasing 
the protein with the protease makes it much cleaner. Most of the protease has a 
His-tag, which allows at the same time to bind it to do beads (of course this 
slows down a bit the cleavage therefore mixing is required). Alternatively, 
GST-(only)-tag protease is better used for the cleavage on Ni beads and then 
captured on the GSH resin.
* 3C protease is much more efficient for cleaving in the presence of detergent, 
there is an extensive study on that topic 
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/)
* Both proteases are easy to produce, as mentioned, and easily 100-300 mg can 
be obtained from 1 L culture (TB or auto-induction with OD= 8-16)
* 3C protease is not happy at high protein concentrations (>4 mg/ml) in low 
salt buffer (150 mM). Therefore huge loss is observed if, during the 
Ni-elution, low salt buffer is used as the protein comes from the Ni-NTA at a 
concentration 15-20 mg/ml. Please use at least 500 mM NaCl for that step or 
better for any step from lysis to elution.
* And yes both proteases have cross-activity and can cleave the opposite target 
site with lower activity.
These are the few points coming from the top of my head.
I hope they are helpful and wish everyone happy cleaving of the fusion proteins 
:)

Kind regards,
Nikolay

Nikolay Dobrev 
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents https://twitter.com/emblevents 
|http://facebook.com/embl.org  | http://youtube.com/user/emblmedia
Visit http://www.embl.org/events  for a complete list of all EMBL events.

> On 12/07/2022 11:51 PM Lior Almagor  wrote:
> 
> 
> In my experience, 3C can partially cut TEV sites as well. If using 
> sequentially, it is best to plan to use the TEV first.
> 
> 
> > > On Dec 7, 2022, at 2:42 PM, Lau Kelvin 
> <5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk 
> mailto:5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk > wrote:
> > I agree. I think it depends on the lab and vectors and personal 
> > preference. 
> > 
> > But David, have you used them sequentially? I have once tried to 
> > make a His-GST-ENLYFQ-3C construct, and I found that it was self cleaving 
> > during expression. However I have never tried to replicate the results in 
> > vitro.
> > 
> > 
> > 
> > -- 
> > Kelvin Lau
> > Protein production and structure core facility - PTPSP
> > EPFL SV PTECH PTPSP 
> > AI 2146 (Bâtiment AI) 
> > Station 19 
> > CH-1015 Lausanne
> > Switzerland
> > Email: kelvin@epfl.ch mailto:kelvin@epfl.ch
> > Phone: +41 21 69 34494
> > 
> > 
> > > > > On 7 Dec 2022, at 21:38, David Briggs 
> > mailto:david.bri...@crick.ac.uk > wrote:
> > > Hi Gloria,
> > > 
> > > Both can be made very easily in E.coli.
> > > Both are active at 4°C, but especially 3C, I think. 
> > > 
> > > I have plasmids for both somewhere in the freezer (you might 
> > > find someone closer to you who can send HRV3C, but 

Re: [ccp4bb] TEV vs HRV3C

[Lior Almagor]

In my experience, 3C can partially cut TEV sites as well. If using 
sequentially, it is best to plan to use the TEV first.

> On Dec 7, 2022, at 2:42 PM, Lau Kelvin 
> <5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> I agree. I think it depends on the lab and vectors and personal preference. 
> 
> But David, have you used them sequentially? I have once tried to make a 
> His-GST-ENLYFQ-3C construct, and I found that it was self cleaving during 
> expression. However I have never tried to replicate the results in vitro.
> 
> 
> 
> -- 
> Kelvin Lau
> Protein production and structure core facility - PTPSP
> EPFL SV PTECH PTPSP 
> AI 2146 (Bâtiment AI) 
> Station 19 
> CH-1015 Lausanne
> Switzerland
> Email: kelvin@epfl.ch 
> Phone: +41 21 69 34494
> 
>> On 7 Dec 2022, at 21:38, David Briggs > > wrote:
>> 
>> Hi Gloria,
>> 
>> Both can be made very easily in E.coli.
>> Both are active at 4°C, but especially 3C, I think. 
>> 
>> I have plasmids for both somewhere in the freezer (you might find someone 
>> closer to you who can send HRV3C, but if you cannot, let me know off list).
>> 
>> I don't see any particular benefit of one over the other, but having both in 
>> your freezer means you can cleave off tags sequentially as needed by your 
>> purification strategies.
>> 
>> HTH,
>> 
>> Dave
>> 
>> Dr David C. Briggs CSci MRSB
>> Principal Laboratory Research Scientist
>> Signalling and Structural Biology Lab
>> The Francis Crick Institute
>> London, UK
>> ==
>> about.me/david_briggs 
>> 
>> From: CCP4 bulletin board > > on behalf of Gloria Borgstahl 
>> mailto:gborgst...@gmail.com>>
>> Sent: Wednesday, 7 December 2022, 20:26
>> To: CCP4BB@JISCMAIL.AC.UK  
>> mailto:CCP4BB@JISCMAIL.AC.UK>>
>> Subject: [ccp4bb] TEV vs HRV3C
>> 
>>  
>> External Sender: Use caution.
>>  
>> Hello my fellow structural biologists,  I am contemplating why some choose 
>> the HRV3C protease site over TEV for their fusion proteins.  Does anyone 
>> know?  Can HRV3C be made easily in homelab?  Does anyone have a plasmid?  
>> Thank you, G
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
>> 
>> The Francis Crick Institute Limited is a registered charity in England and 
>> Wales no. 1140062 and a company registered in England and Wales no. 
>> 06885462, with its registered office at 1 Midland Road London NW1 1AT
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
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> 
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Re: [ccp4bb] TEV vs HRV3C

[Lau Kelvin]

I agree. I think it depends on the lab and vectors and personal preference.

But David, have you used them sequentially? I have once tried to make a 
His-GST-ENLYFQ-3C construct, and I found that it was self cleaving during 
expression. However I have never tried to replicate the results in vitro.



--
Kelvin Lau
Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 34494

On 7 Dec 2022, at 21:38, David Briggs 
mailto:david.bri...@crick.ac.uk>> wrote:

Hi Gloria,

Both can be made very easily in E.coli.
Both are active at 4°C, but especially 3C, I think.

I have plasmids for both somewhere in the freezer (you might find someone 
closer to you who can send HRV3C, but if you cannot, let me know off list).

I don't see any particular benefit of one over the other, but having both in 
your freezer means you can cleave off tags sequentially as needed by your 
purification strategies.

HTH,

Dave

Dr David C. Briggs CSci MRSB
Principal Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Gloria Borgstahl 
mailto:gborgst...@gmail.com>>
Sent: Wednesday, 7 December 2022, 20:26
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] TEV vs HRV3C


External Sender: Use caution.

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G



To unsubscribe from the CCP4BB list, click the following link:
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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] TEV vs HRV3C

[Chun Luo]

HRV3C cut of an N-terminal tag usually leaves GP at the N-terminus. TEV usually 
leaves G/S. Structural biologists may prefer TEV. However, HRV3C has more 
robust activity than TEV. HRV3C is a better choice for removal of C-terminal 
tags.

 

To save precious time, Accleagen has Turbo3C 
(http://www.accelagen.com/Turbo3C.htm) available at a reasonable price. It has 
dual GST- and His-tags for easy removal. We have other enzymes for protein 
purification.

 

Chun Luo

Accelagen

 

 

From: CCP4 bulletin board  On Behalf Of Gloria Borgstahl
Sent: Wednesday, December 7, 2022 12:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] TEV vs HRV3C

 

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G

 

  _  

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 =1 




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Re: [ccp4bb] TEV vs HRV3C

[Cyprian Cukier]

I believe HRV3C might be more efficient if you need to cleave in the absence of 
reducing agents (e.g. extracellular proteins).

Best regards,
Cyprian

Cyprian Cukier, PhD
Selvita S.A.

From: CCP4 bulletin board  On Behalf Of Crissy L Tarver
Sent: Wednesday, December 7, 2022 9:38 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] TEV vs HRV3C

We use the HRV3C protease site frequently in our lab. It provides better 
on-bead/column cleavage than TEV. We have a plasmid as we make 3C protease in 
our lab and keep stocks in our -80C. I can send you our protocol if you wish.

Crissy

Crissy L Tarver, PhD
Department of Structural Biology
Stanford University School of Medicine


Confidentiality notice: This communication and any attachments may contain 
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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Gloria Borgstahl 
mailto:gborgst...@gmail.com>>
Sent: Wednesday, December 7, 2022 12:26 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] TEV vs HRV3C

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G



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Re: [ccp4bb] TEV vs HRV3C

[Crissy L Tarver]

We use the HRV3C protease site frequently in our lab. It provides better 
on-bead/column cleavage than TEV. We have a plasmid as we make 3C protease in 
our lab and keep stocks in our -80C. I can send you our protocol if you wish.

Crissy

Crissy L Tarver, PhD
Department of Structural Biology
Stanford University School of Medicine


Confidentiality notice: This communication and any attachments may contain 
confidential or privileged information for the use by the designated 
recipient(s) named above. If you are not the intended recipient, you are hereby 
notified that you have received this communication in error and that any 
review, disclosure, dissemination, distribution or copying of it or the 
attachments is strictly prohibited. If you have received this communication in 
error, please contact me and destroy all copies of the communication and 
attachments. Thank you.

From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Wednesday, December 7, 2022 12:26 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] TEV vs HRV3C

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G



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Re: [ccp4bb] TEV vs HRV3C

[David Briggs]

Hi Gloria,

Both can be made very easily in E.coli.
Both are active at 4°C, but especially 3C, I think.

I have plasmids for both somewhere in the freezer (you might find someone 
closer to you who can send HRV3C, but if you cannot, let me know off list).

I don't see any particular benefit of one over the other, but having both in 
your freezer means you can cleave off tags sequentially as needed by your 
purification strategies.

HTH,

Dave


Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Wednesday, 7 December 2022, 20:26
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] TEV vs HRV3C


External Sender: Use caution.

Hello my fellow structural biologists,  I am contemplating why some choose the 
HRV3C protease site over TEV for their fusion proteins.  Does anyone know?  Can 
HRV3C be made easily in homelab?  Does anyone have a plasmid?  Thank you, G



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