I will soon start expressing GFP-quimeric proteins in E. coli (BL21) and then purifying them using a 6xHIS tag. Does anybody have experience working with recombinant GFP proteins it complicated? Do they tend to form inclusion bodies? Do I have to work always on Dark?. Any comments and advices will be greatly appreciated.
In our lab, we routinely use the GFP-Trap from Chromotek to purify GFP-tagged proteins from mammalian and E. coli cells. You will find that it is far more efficient and cleaner than using His-tag for purification.
http://www.chromotek.com/products/nano-traps/gfp-trap/
I have had experience in purifying various forms of eGFP and I have never had any issues in purifying it via the His Tag on Ni-NTA resin. GFP is wonderfully stable in comparison to other proteins I have used (eg. luciferase) so you should be fine.
With regards to working in the dark, you only have to work in the dark if you have incorporated anything light sensitive into your particular chimera, a phenyl azide etc since the GFP alone is not sensitive to light.
http://www.cell.com/structure/pdf/S0969-2126(14)00115-4.pdf
I hope this helps.
It's really easy. It's green. So you can exactly see if it's being made, if it's active and where it's going. Even the cell pellet is green!
I have purified several His-tagged, GFP fusion proteins, and I agree it should be very easy - depending on what your fusion protein is!. GFP does not require you to work in the dark. As well as making your fusion protein visible to the naked eye so you can track it throughout purification, GFP is a very soluble protein, and will hopefully stabilise your fusion protein. You can also use the GFP flourescence to quantify your protein more accurately than absorption at 280nm. The pI of GFP is ~6, so one piece of advice I would give (which applies to all protein purification). Would be to see how this influences the pI of your entire fusion protein. Check the pI of your entire fusion protein (use an online tool such as protparam), and use a purification buffer with an appropriate pH. Although you can use a GFPtrap, it requires fairly harsh conditions to elute the protein, so bear this in mind. If you are using NiNTA, make sure you use a pH of >7. The purification should be relatively clean if you use an appropriate loading/washing concentration of imidazole, and wash the NiNTA with enough wash buffer.
Thank you very much for all the answers! Let´s hope my fusion proteins behave!
@ Andrew N Blackford This might be a stupid question. How do you elute your GFP tagged protein from the beads? My protein has eGFP tagged.
Abir Chakraborty depends on what you are eluting your protein for. If you are doing IPs from cell lysates, then you can elute with standard 2X Laemmli buffer. If you want to preserve you protein's activity, you could try to elute with glycine (protocol here: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/m8823bul.pdf). If that doesn't work, you could clone in a cleavage site for a protease between your GFP tag and protein of interest to elute it, if you don't mind losing the tag.
Thanks for your reply. I would like to use that protein as labelled protein. My plan is to add endotoxin free protein directly in cells.Is pH 2.2 good for protein's native structure ? Do I need to neutralise the pH immediately after purification ?
Abir Chakraborty yes, you need to neutralise with 1 M Tris pH 8.0, see the link from Sigma in my previous answer. Then you could buffer exchange afterwards to get your protein into a different buffer for your next application.
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