Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Flavio Di Pisa]

Dear all,
thank you for your valuable suggestions.

Have a good day,
Flavio.

Il 16/02/24 15:36, Karla J. F. Satchell ha scritto:


Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide 
info on one type recommended by another. Others may have further 
suggestions for Flavio.


*From: *CCP4 bulletin board  on behalf of Karla 
J. F. Satchell 

*Date: *Friday, February 16, 2024 at 7:13 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi everyone—

Thought I might chime in.

This technology was developed in my lab based on our studies of toxins 
and we have methods papers and two patent.


The CPD-tag works great for production of protein with minimal 
residual residues on the protein. Our studies indicate that it can 
help solubilize protein during expression. Protein is purified with 
the C-terminal CPD tag, and the entire CPD and 6xHis-tag are released 
by addition of InsP6 (phytic acid) which is very inexpensive.


The only restriction of the recognition sequence is small-Leu-small so 
a small residue plus Leucine does remain on your protein. There does 
also need to be a little flexibility at the end of your protein for 
access to the protease so our vectors add extra Gly-Ala so the 
residual is GAAL. CPD is particularly useful for small proteins if the 
additional residues do not interfere with your assays.


https://pubmed.ncbi.nlm.nih.gov/28056928/ 



https://pubmed.ncbi.nlm.nih.gov/31773580/ 



https://patents.google.com/patent/US8257946B2/en 



https://patents.google.com/patent/US8383400B2/en 



Here is a link to the original paper on mechanism of action of CPD

https://pubmed.ncbi.nlm.nih.gov/19620709/ 



These are not commercially available as despite multiple marketing 
attempts, there was little interest mid-2010s from biotech for 
licensing new cloning technologies and we abandoned advanced development


If this technology is interesting to you to add to your commercial 
biotech portfolio, please feel free to reach out to me.


Flavio, we can share our vector, but my institution does require an 
MTA as technology is patented. Most people just use synthetic DNA to 
generate the exact clone they want so we rarely get requests but happy 
to share sequences if you need for your synthetic design.


Karla Satchell

*From: *CCP4 bulletin board  on behalf of 
Srivastava, Dhiraj 

*Date: *Friday, February 16, 2024 at 6:42 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi Flavio

      While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease 
cleavage site of your choice. However this strategy will results in 
quite a few extra residues from protease site.


An alternative, which is not commercial but available through addgene, 
is cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in 
solubility and expression.


https://www.addgene.org/38251/ 



There are other variants of this vector available on addgene that you 
can choose from. Depending on your c terminal sequence, it may leave 
either no extra residues or only one or two residues.


Dhiraj



*From:*CCP4 bulletin board  on behalf of Flavio 
Di Pisa 

*Sent:* Friday, February 16, 2024 3:33 AM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the 

Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide info on 
one type recommended by another. Others may have further suggestions for Flavio.

From: CCP4 bulletin board  on behalf of Karla J. F. 
Satchell 
Date: Friday, February 16, 2024 at 7:13 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/
https://pubmed.ncbi.nlm.nih.gov/31773580/

https://patents.google.com/patent/US8257946B2/en
https://patents.google.com/patent/US8383400B2/en

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.

Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/
https://pubmed.ncbi.nlm.nih.gov/31773580/

https://patents.google.com/patent/US8257946B2/en
https://patents.google.com/patent/US8383400B2/en

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



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Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Srivastava, Dhiraj]

Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



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/



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/