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 <CCP4BB@JISCMAIL.AC.UK> on behalf of Karla
J. F. Satchell <k-satch...@northwestern.edu>
*Date: *Friday, February 16, 2024 at 7:13 AM
*To: *CCP4BB@JISCMAIL.AC.UK <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://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/28056928/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HimFkBThs$>
https://pubmed.ncbi.nlm.nih.gov/31773580/
<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/31773580/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HijC4KPFp$>
https://patents.google.com/patent/US8257946B2/en
<https://urldefense.com/v3/__https:/patents.google.com/patent/US8257946B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HirGMggut$>
https://patents.google.com/patent/US8383400B2/en
<https://urldefense.com/v3/__https:/patents.google.com/patent/US8383400B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HiqMuRtc4$>
Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/
<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/19620709/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HinAiY8dQ$>
These are not commercially available as despite multiple marketing
attempts, there was little interest mid-2010s from biotech for
licensing new cloning technologies and we abandoned advanced development
If this technology is interesting to you to add to your commercial
biotech portfolio, please feel free to reach out to me.
Flavio, we can share our vector, but my institution does require an
MTA as technology is patented. Most people just use synthetic DNA to
generate the exact clone they want so we rarely get requests but happy
to share sequences if you need for your synthetic design.
Karla Satchell
*From: *CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of
Srivastava, Dhiraj <0000bda2be8a675a-dmarc-requ...@jiscmail.ac.uk>
*Date: *Friday, February 16, 2024 at 6:42 AM
*To: *CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid
expression vector for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c
terminal tag, you can easily make your own by introducing protease
cleavage site of your choice. However this strategy will results in
quite a few extra residues from protease site.
An alternative, which is not commercial but available through addgene,
is cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in
solubility and expression.
https://www.addgene.org/38251/
<https://urldefense.com/v3/__https:/www.addgene.org/38251/__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdL2xUs9Cg$>
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 <CCP4BB@JISCMAIL.AC.UK> on behalf of Flavio
Di Pisa <dipi...@gmail.com>
*Sent:* Friday, February 16, 2024 3:33 AM
*To:* CCP4BB@JISCMAIL.AC.UK <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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