Why GFP gets cutted from my fusion protein? - (Sep/04/2012 )

Hi everyone!

I've got a problem:

after around two weeks of fusion protein expression (protein-Cterminus-Thrombin_tag-EGFP-6xHis_tag) I see 2 bands on a gel (instead of one band). These bands correspond to the protein and GFP tag (last one is fluorescent on the gel).

In my elution buffer I have protease inhibitor, 1M imidazole, 50 mM TRIS-HCl(pH 7.6), and 500 mM NaCl.

Why does it happens?

Sorry if it's already has been discussed before, but I didn't find it yet.

Thank you for your suggestions,

Cheers

How big is your protein?

Hi,
My protein is 32 kDa

Does your protein also express by itself in a soluble manner?

And forgot to ask you the most important question: what expression system are you using? (E coli??)

Hi,

This is the membrane protein. I forgot to mention, - 0.15 mM of DDM detergent presents. Expression system is Yeast (INVSc).

I believe (with big stress on I) that the yeast in not very happy with expressing your protein. Even though you are close to have it almost expressed. I mean: you do everything right: you express a multidomain (fused proteins is like multidomain protein) in a higher than bacteria organism. In theory, yeast should deal nicely with your construct in terms of size of protein and complexity. On the other hand: transmembrane protein --> ouch. I would try other types of expression yeasts or other media (minimal media or there is this cool ethanol based media) or other expression plasmids with milder promoters. I believe that you are using the GAL one which is a bit too strong. There are also constitutive promoters in yeast (even though these plasmids are not sold anymore but some people might still have them). Usually playing with the media and promoters and temperatures is enough for expression, but transmembrane protein... maybe you consider even changing the organism. I would go for insect cells, but this is not for every lab because if you don't have it set up, you can't do it.

To come back to what I believe that happens: when cells are not happy over-expressing a protein they either express it aggregated or degraded to its stable domains.

Andreea

Could it be that your protein is cleaved at the C-Terminal domain? Have you check if there is any cleavage site at the C-terminus? We had a very similar problem tagging our protein with c-myc and after several trials we found that the C-terminal domain of our protein had a sequence (CRR) that apparently is cleavage by a protease to make our protein functional. The consequence was clear... we were loosing our tag, not the protein!!!

Situation is like:

protein expression => purification => gel at the same day - one fat band => same sampe two weeks later - 2 bands (!!??!!) something is cutting it, but what and how to prevent it is the question

ascacioc on Thu Sep 6 15:22:25 2012 said:

How do you store your protein? What you say is a common situation. Especially if you store it at 4oC. I usually snap freeze it in liquid nitrogen and then store it at -80 until next use. But for the first trial: I snap freeze only an aliquot of the protein, I thaw it and check whether it precipitates or smth is fishy. Usually it doesn't. But if it does, you can add glycerol or other things that make the protein more comfortable and freeze it with those additives. I have seen proteins self-cleaving when in fridge (faster on the bench) and it is a mystery (at least to me) what in the solution except themselves makes them cleave. But it is how it is. Sometimes it does that while frozen. You usually lose some by freeze-thawing (centrifuge at max for 10 min on table top centrifuge at 4oC and take the supernatant; aggregates will be on the bottom), but there is always smth you get back.

Andreea