Hi everyone,
I have to express different mammalian proteins tagged with different fluorophores (such as GFP, YFP and others) under bacterial expression system and to use these purified proteins for FRET studies in vitro.
I wonder if florophores expressed in this system would be active? will I be able to directly use the purified proteins for FRET, are they remain active or need to go through refolding steps here??
Thank you.
Dear Ashish
i expressed and purified gfp from e.coli many times and it is fluorescent. You can see a nice picture of cell pellet expressing gfp
at minute 3 of ProteoCool n° 5 on my blog: https://proteocool.blogspot.com/p/cloning.html?m=1
The only suggestion is to perform induction at 25°C because at higher temperatures the expression rate is too high and not al the protein expressed is well folded and fluorescent and once purified it was stable and fluorescent at 4°C for years.
i never tried yfp and rfp but i suspect that is the same.
good luck
Manuele
Ashish Kumar
Hi, Ashish! I have recently purified GFP-His tagged protein in BL21 (DE3). The recombinantly purified GFP His tag protein is fully active as confirmed by checking the spectral properties. In case you would want me to elaborate more or share the data with you, I would be happy to do that.
As mentioned by the other answerers, at least GFP and YFP should be fine in E. coli (not sure about RFP, but mCherry works). However, if you are using standard GFP, the fluorophore takes a relatively long time to mature in E. coli. You should consider using an enhanced variant or better yet the superfolder variant of GFP for faster fluorophore maturation.
Thanks everyone.!
From your experiences it sounds like expression of active fluorophore tags are possible.
Considering your inputs, I am thinking to induce protein synthesis at 16'C-20'C. If you could also suggest any other points or possibilities, please do that.
I will update on my experiences soon.
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