Hi,
I have cloned my protein with TEV protease site (His tag-TEV site-linker-protein). I purify my protein with Ni-NTA and then do on column TEV cleavage (overnight). I hardly see any TEV cleavage.
1. my protein is a membrane protein, so I have detergents in the buffer (tried TEV cleavage with DDM and LDAO containing buffers), but considering a low TEV activity in the presence of detergents if at all, even if I increase the amount of TEV, there is hardly any on column TEV cleavage.
2. For my protein construct, I have made 2 constructs with 5 and 12 amino acid linker between TEV site and my protein, but none of them give on column TEV cleavage
3. Being a membrane protein, the yield is very low, and thats why I want to go for on column TEV cleavage, to reduce the no. of columns and thus, the loss of protein with every column
4. TEV is as per the 2009 paper (PMID: 18988033), wherein to make it compatible with Ni-NTA, I have changed His tag to GST tag. So, my TEV has GST tag at N-terminus and a (arginine)5 tag at C-terminus.
5. I have tried elution of my protein from Ni-NTA, diluting it to reduce imidazole and then do TEV cleavage, it works comparatively better but elution from ni-nta is very dirty, and concentrating my protein with amicon concentrator (tried diff KDa taking into account detergent micelle size) produces a viscous solution which appears as a smear on the sds-gel.
6. in one trial of on-column TEV cleavage, it appears that might be TEV is binding non-specifically to Ni-NTA. Is it possible with an (arginine)5- tag?
7. Since Ni-NTA is not compatible with EDTA and DTT which are reqd for TEV activity, as suggested online, I had supplemented the buffer with reduced glutathione and sodium citrate for TEV activity, but still no improvement.
Could anyone suggest anything for TEV on-column cleavage?
Dear Megha Abbey,
TEV is doing the cleavage much faster, when the reaction partners are in solution, and not on solid phase. So we would recommend to purify on INDIGO Ni, dialysis of the eluate gaginst TEV buffer, TEV cleavage, and removal of his and TEV (which should be his-tagged) by INDIGO, which is an IMAC resin, compatible with high concentrations of EDTA and DTT. Now you'll find your protein of interest in the flow-through.
More information about INDIGO you can find at
https://cube-biotech.com/products/purification-resins/his-affinity-resins/100-indigo-ni-agarose/
Best, Roland
Dear Megha Abbey,
Tev requires a reductive environment to work properly, and NI-columns are no very suited for that matter. What I do is past Ni-affinity chromatography and dialyzes, digest my protein with TEV supplemented with GSH and GSSG in small concentrations and the results are just fine. They will provide enough reductive potential for TEV to work properly.
I would try elution of your protein from NI-NTA , dialyzes, TEV digestion coupled with GSH and GSSG, centrifugation and only then amicon concentration. Maybe the viscous aspect of your concentrated protein is over-concentration, perhaps? But I wouldn’t recommend reductive agents on the column on high concentrations. Also, are you sure your TEV (if recombinant) does not have a HIS-tag? Hope I could help you. Best luck.
We have used TEV for years and whilst it is a nice simple purification and potential quick and easy cleavage there are numerous factors that impact on its ability. Generally speaking the best we have achieved is complete TEV cleavage after 2 hrs at RT with a somewhat concentrated protein ie if you elute in 30 mL then conc it down to 5mL and add TEV. Also it can work anywhere from 1:100 dilution to 1:1 dilution and unfortunately 1:1 is what often works for membrane proteins.
I would also recommend doing it in solution as on resin adds all sorts of steric issues to the mix. Sometimes it may work but alot of the time it doesnt from my experience. Buffer composition will also effect it as low or high pH, high salt or imidazole will effect its cleavage efficiency.
For a nice run down...
http://wolfson.huji.ac.il/expression/local/tev.pdf
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