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: ----------- Charlie (ucbc...@ucl.ac.uk) 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. ----------- ----------- Allan (a.p...@qmul.ac.uk) 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? ----------- ----------- Avinash (avinashkal...@gmail.com) 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. ----------- ----------- Naoki (nsa...@sci.hokudai.ac.jp) 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. ----------- ----------- Chun (c...@accelagen.com) You may check the protocol at our TurboTEV website (http://www.accelagen.com/TurboTEV-protocol.htm). It should be very simple. ----------- ----------- Rajendra (rajendr...@gmail.com) 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. ----------- ----------- Bart (bart.ha...@ualberta.ca) 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. ----------- ----------- Ralf (jau...@gis.a-star.edu.sg) 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. ----------- ----------- Kirsty (k.l...@exeter.ac.uk) 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. ----------- ----------- James (jver...@virginia.edu) 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. ----------- ----------- Marcus (marcus.fisl...@vib-vub.be) 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. ----------- Thanks Ronaldo.
