Hello everybody, I have been expressing proteins that contain disulfide bridges and I usually use pET32 which cantains Trx as chaperone and Rosetta gami as strain. This system works for some proteins but not for others. Sometimes I get enough protein in the supernatant, in other cases I got all the protein in the pellet. I don't want to do refolding because i lose much protein in the process. Do you know other system that I can try?
Hi Patricia,
Every protein is different hence you may need to trouble-shoot expression issues. There are a few things to take in to consideration (I'm assuming that the protein is foreign to E. coli?)
1. Try different growth conditions: i.e. induction time length (sample over a number of hours post induction), temperature (lower temp helps with protein folding), media (try different media: 2xYT, LB, Teriffic broth). TB is pH buffered and may help with expression in some cases.
2. Plasmid/promoter: T7 is a powerful promoter and may not be suitable for expression of all proteins. Use a weaker promoter, we had excellent results with pTrc (invitrogen) for soluble protien.
3. Even try a different strain of bacteria.
4. As suggested by Garitsa you could try periplasmic expression
5. You could try a codon biased strain i.e. RIPL
good luck
You can also try Shuffle cells that might prove better than Rosetta gami - but of course, every protein is different...
The Trx tag is thioredoxin that reduces other proteins by cysteine thiol disulfide exchange. This is possibly not the best idea if you want to conserve disulfide bridges in your expressed protein.
It will be essential for you to use a bacterial strain that has a TrxB (thioredoxin reductase) mutation such as Origami, Rosetta-Gami or (my favorite) AD494.
Also, to maintain folding and solubility, you must express at low temperature (25 °C or lower).
I have had best results using the vector pCOLD that uses the "Trigger-factor" solubilizing tag and a "cold-shock" promotor. Therefore, induction on ice and production over-night at 16°C.
Hello Garitsa:
Thanks for the suggestion, I used pET 22 to send recombinant protein to periplasmic space, but did not work, because as Ian said, every protein is different.
Thanks Ian R Wilkinson for all suggestions you made. I have tried many things you mentioned, but I guess each protein has to be tested in different conditions. The weak promoter is a good idea, unfortunatelly I don´t have any vector with that promoter right now.
Hello Ian Robbins, Roseta gammi has the TrxB mutation, but as i mentioned, some proteins are produced just fine, but anothers don't. I always express at low temperature, usually 25 degrees. I haven't used pCOLD vector, but sounds like a good idea to me, I will read about it and I will try to get the vector and tested it. Thanks for all the suggestions.
Ondřej Vaněk, I have tried both. But as you said every protein is different. Thanks, good day.
If it is not a secret, could you share the name of the protein and the purposed downstream application you want the protein for? We are all painfully aware that all proteins are different, but the knowledge of what are we talking about could be useful.
I am experimenting with something new that might be useful.
Hello,
you may try also to produce the recombinant protein in the SHuffle T7 E. coli strain (see the New Englands Biolabs website) or SHuffle T7 Express lysY if your protein is toxic and do not cultivate at 37°C but at 28° or even at lower temperature for a longer time,
good luck,
Colette Duez
Hello Dario, I'm working with defensins from C. limpidus in order to do MNR and cell toxicity studies. I'm not able to obtain enough quantities to do that because all the agreggation issues.
Patricia
I recently published the following article
http://www.nature.com/ncomms/2015/150408/ncomms7826/full/ncomms7826.html
This research uses a new way of solubilizing membrane proteins in vivo. Using this technique we are using the disulfide bond machinery of E coli (DsbA, DsbB, DsbC) so produce disulfide bonds in the cytosol of E coli. In some cases is comparable or better than Shuffle cells. By looking at defensins they could be a great target for my system. Also the system described in the paper could be used to express hard to express proteins. It seems that some defensins are amphipatic or can form pores.
It could be exciting to to use my system to improve yours. Let me know if you would like to establish a collaboration
I noticed also these proteins are not very easy to handle. In this paper
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC520777/
they express and purify defensins from yeast (P pastoris). This could also be an alternative for you
Thanks Dario, I will review your paper to see if I can use your system.
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