Hi there,

Cleavage site accessibility should be improved using non denaturing concentration of detergent or of urea (up to 2M).

Dear Selene Casella

in my experience when the MBP cleavage show low efficiency could be do to the poor solubility and/or high aggregation propensity of the specific protein that lead to the formation of a sort of soluble micelles where the protein is buried inside and the TEV accessibility to the cleavage site si limited to MBP steric hydrance.

Did you try to load in a SEC coloumn the purified MBP-protein fusion to see if it is monomeric and/or aggregate?

Ciao

Manuele

Hi Dominique,

I've tried with 0.5% tritox X-100 but the results are just slightly better. Do you recommend to try urea? I need the protein for activity assay later on.

Dear Manuele Martinelli,

yes I tried SEC column. When I run my sample, I have a peak at the similar elution volume (40-43ml) of the first peak (45ml)of the STD (which could be aggregated?). The standard I use (biorad 151-1901) has 7 peaks but the first (45ml) and the last are not related to the std proteins and I think they are big and small aggregates. Do you confirm? When I load this fraction (the one should be big aggregates)in SDS page, I see the band of my tagged protein together with another protein (which I know interact with it). However, If I assume that this peak is made of aggregates is quite small compared to the peak of the purified protein (less than 1/10 in volume by eye). Do you I also know if the MBP tag could enhance agglomeration between proteins and how to avoid it?

I like the answers regarding the triton and urea, but to get the best cleavage efficiency I can recommend to re-design the vector, something like: MBP-linker_sequence_TEV_protein. MBP is big and likes to form soluble aggregates. The TEV protease needs free access..MBP is nice to work with, but sometimes cleavage is a lottery. Good luck!

Thanks Petr Rathner , I think I'll start to consider more seriously a change of tag (or addition of a linker). Can you recommend a paper that speaks about MBP aggregates? Just curiosity since I also built a mutant of my protein that should not make dimers and it is still partially in dimers (I was thinking if it was the fault of the tag).

you will find a lot info about MBP and aggregation (also on researchgate). The problem is that the targed proteins maybe interacts with the MBP. MBP is known to be sticky. I have good experience with GST tag. Easy to purify and cleavage was also ok. Just for fun, can you post your protein sequence and the mutation position? (I understand if confidental). BR

The problem is most probably not MBP itself but your vector design. When you are redesigning your vector, consider including couple more residues right after the TEV cleavage site. If TEV site is directly adjacent to your protein of interest, it could hinder cleavage efficiency. MBP-Linker-TEV site (ENLYFQG) POI works for me 95% of a time. You could use NEB Q5 Site directed mutagenesis to insert couple residues hassle-free. Hope that helps.

Dear Burak Gulen , you suggestion is quite right. However, I was hoping to find an alternative solution before to make all my constructs again. Which are a lot...

I think you do not have many choices. Either you should accept the poor cleavage efficiency and purify your protein by recovering as much as possible or reclone. I been there. Recloning, expression and purification seem a lot of work but it might save you time in the long run. If you do not need tons of protein in the first place, 40% cleavage could be enough too. You can still get a lot of protein to work with. If your purpose is to increase the cleavage efficiency, changing conditions won’t help much. TEV protease needs its space to cut and I think only option is to reclone your construct.

Thank you all. Here how I've solved the problem. It looks like that changing the purification steps improves the removal of the MBP tag (the tag was almost completely removed using same concentration of TEV used previously). We suppose that after protein expression, a step of dialysis overnight at 4C, gives time to the proteins to reach the correct folding. Larger aggregates were also removed by affinity column. This helps the action of TEV protease.

Hi Selene, I am interested about the solution because I noticed the same issue. Could you explain me better how you solved the problem? Do you perform an o.n. dialysis of supernatant before any column? Thank you in advance for your precious help

Hello Raffaele Sabbatella Raffaele, I broke the cells, then used MBP-trap beads to purify my tagged protein in the supernatant. After, the protein was dialyzed overnight. Then performed a purification in an affinity column. After all this, the protein is nicely cut by the TEV protease. Note that the affinity column you need, depends on the characteristics of your protein. Therefore, you need to try a few methods to see which one traps your protein better. Also, dialysis will help you decrease the salt and remove the maltose. I really hope this will help you. Let me know.