Dear CCP4bb users (this the summary of an off-topic question),
First of all I would like to thank all users who answered my previous question.
I compiled a summary of all answers I received in the last week.
The original question was:
> We have a few proteins being expressed as HIS-TAG_(TEV_cleavage_site)_PROTEIN
and we are about to initiate the purification steps. We have already used the
HiTrap-Chelating columns from GE for the first purification step (affinity
chromatography) and we would like to move forward with TEV digestion and a
second purification step. We know these following steps are very
protein-dependent, but we were wondering one could share his/her experience in
the following steps: removal of imidazole, cleavage protocol and cleavage
identification, second chromatography, etc.
And the answers were:
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Charlie ([log in to unmask])
Limitless suggestions, but as you are about to embark on TEV digestion, one
thing you can do is do this within a dialysis tube and 1:20 and dialyse for the
magic period (overnight) to get rid of the imidazole, then follow this with an
IMAC step to remove uncleaved protein and the TEV.
Remember to equilibrate your IMAC column including a little imidazole 5-30mM
because, despite your wanting your tagged stuff to stick, some proteins stick to
the resin anyway if you don't equilibrate. To make doubly sure of this I always
elute off the beads and run it on a gel, just to see what is going on.
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Allan ([log in to unmask])
I would advise you to actually read the "manual" of the TEV where you bought it
from. They usually provide you with the list of info on which buffer you should
use. This will help you to identify if there is a need for you to remove
imidazole, high salt, or change buffer.
You can easily remove imidazole via dialysis but I personally don't trust
dialysis, don't like them as it can possibly degrade your protein. I think
there are desalting column that exist that can help you buffer exchange.
Otherwise, go ahead with your digestion even with imidazole (that is, if TEV is
not inhibited by imidazole).
Usually proteins can be crystallized with HIS tag on, so if crystallization is
your main objective, why not try crystallizing first with tag on?
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Avinash ([log in to unmask])
I was wondering why you don't consider the option of on-column cleavage in your
case. what i will be tempted to do is to dissolve the the required amount of TEV
in 1.5 column volumes (preferably 1CV) of of the buffer (with no imidazole) and
pass through the column with bound protein of your interest... i would then let
the column stand for the a desired duration so as to let the TEV protease to
finish its job. by closing both ends of the column (this prevents and drying of
the resin)... then just pass the wash buffer (with no imidazole) and collect the
flow through to get your cleaved protein, nicely seperated from His6-tag and TEV
protease.
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Naoki ([log in to unmask])
During TEV cleavage, DTT and EDTA are needed. TEV protease is a cystein
protease, that is reason why adding reducer.
1mM DTT and 0.5mM EDTA are first choice.
5mM 2-ME, and glutathione (3mM reduced, 0.3mM oxidized) are alternative reducer.
I usually use TEV in following buffer condition, for overnight reaction at RT.
20mM Tris-HCl pH8.0
150mM NaCl
1mM DTT
1mM EDTA
After the first HisTrap purification, you should remove imidazole and change
buffer to the above, using desalting column or dialysis. In my experience,
imidazole inhibits the cleavage activity of TEV.
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Chun ([log in to unmask])
You may check the protocol at our TurboTEV website
(http://www.accelagen.com/TurboTEV-protocol.htm). It should be very simple.
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Rajendra ([log in to unmask])
I think this should be very straight forward, Generally, after Ni-column protein
can be digested with TEV (1:50, or 1:100 ratio) either at room temperature or at
4C depending on the stability of the protein. I had tried this at room
temperature, overnight cleavage at 1:100 (TEV: Substrate) ratio. After TEV
digest you can load it onto the His-trap column again by reducing the
concentration of Imidazole by half or 1/3rd of the original concentration at
which protein was eluted. TEV digested protein will remain unbound to His-trap
column and will be in flow through. TEV with his tag will remain bound to
Ni-column. Finally Imidazole can be removed during the concentration process by
buffer exchange.
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Bart ([log in to unmask])
One thing we often do is to load and wash in high-salt as most protocols
suggest but instead of eluting at high-salt we do a few more column washes at
low salt before eluting with imidazole at low salt. That way you can take your
eluent and load it directly on an ion exchange column without need to do a
buffer replacement first. The only requirement is that the IEX cannot be run at
low pH because imidazole would become protonated and act like a salt.
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Ralf ([log in to unmask])
We immediately desalt after His-Trap (desalting column connected in series) into
an Imidazol free and low salt buffer and digest with 1:50 to 1:100 TEV:substrate
ratios (w:w) at RT or in the cold room (time often a compromise between protein
precipitation and completion of the cut) before proceeding to ion exchange and
size exclusion chromatography.
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Kirsty ([log in to unmask])
After Ni-purification I normally dialyse my fractions into TEV protease cleavage
buffer overnight. I then perform the cleavage and check for completion by
SDS-PAGE. The TEV protease I use (Promega ProTEV) has a HIS tag, so I then pass
the cleaved sample back through a Ni-column, removing the tag and the TEV
protease. I then follow this with a gel filtration step to clean up the
protein.
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James ([log in to unmask])
I would suggest doing the TEV cleavage on column... This was the impurities that
are found with IMAC resin will stay on the column and your cleaved protein will
be released. No need to worry about removing the imidazole since there won't be
any. Then I would recommend a Gel Filtration as a
final step.
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Marcus ([log in to unmask])
i have the experience that it is best to digest with TEV already on the column.
Since in that case you can seperate afterwards the digested proteins from the
proteins still containing the tag and the tag itself. Then I always dialysed my
elution containing imidazole against imidazole free buffer. My favourite second
step is a gelfiltration (size exclusion chromatograhy) which would directly
remove the small fraction of proteinmultimers which might interfere e.g. with
chrystallization. Another possibiloty of course would be ion-exchange
chromatography if your protein has a suitable pI value.
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Thanks
Ronaldo.
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