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
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list of all EMBL events.
On 12/07/2022 11:51 PM Lior Almagor <lior.alma...@gmail.com> 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
<00005aaf8435dbef-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 <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 <http://about.me/david_briggs>
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*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of
Gloria Borgstahl <gborgst...@gmail.com>
*Sent:* Wednesday, 7 December 2022, 20:26
*To:* CCP4BB@JISCMAIL.AC.UK <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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