A friend of mine is getting ready to do some experiments involving purified human proteins expressed in E. coli, and she asked me if I knew anything about protein misfolding - apparently, proteins sometimes misfold when expressed in foreign vectors such as E. coli. Unfortunately, I didn't, but a Google search hit brought up an explanation that's really not that surprising when you think about it, and has to do with the fact that many proteins fold correctly only with the help of chaperone proteins or cofactors. Obviously, this can be a big problem for an experimentalist who wants to get usable amounts of a specific, correctly folded protein.
Does anyone know where to find good information about this problem or have suggestions for how to get around it (with or without changing vectors - I'm not sure if E.coli is a crucial part of the study or not)? The document I linked has some solutions but I'm wondering if there are any resources or "easy" tips out there I can forward along.
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Depends a bit, is it real misfolding or just not expressing properly. The things we generally try, in order of preference (and ease of use)
Rosetta2 pLysS cells (commercial solution, there are other things like Origami and others that are available. They provide things like more of the underused tRNA codons or change redox potential and other things)
Induce at 20 deg C. Then be very patient as your cells take their time to g r o w.
Use autoinduction if you are using a pET vector (Studier paper in Prot Exp and Pur in 2005, don't have the reference to hand)
Shift to a different induction system.
Any combination of the above. Iterate until either a) it works or b) supervisor gives up and suggests trying another protein.
Those are the simple approaches. After that it gets more complicated (find similar proteins, modify somehow, attach fusion partners etc etc)
There are a couple of other approaches that I have had success with over the years.
1) Let it misfold. In E. coli, misfolded proteins are found in inclusion bodies which are actually quite easy to separate away from the soluble proteins. Then you can take the purified inclusion bodies and attempt to refold the protein in solution (i.e. use denaturants like urea and dialyze them out). With the right conditions, you end up with properly folded proteins that can be quite pure.
2) Attach a secretion signal to the 5' end of the gene sequence. This instructs the bacteria to move the protein across the cell membrane, often before it has a chance to misfold. The signal sequence will be clipped off and the protein will be placed in the periplasmic space. Not only is the redox potential of the periplasmic space conducive to formation of disulfide binds, but the protein can be quickly isolated by an osmotic shock.
Yep, those are both things that we haven't often tried (except for HIV protease where refolding works a treat) but have often worked for other people. Moral of the story is that it is usually a painful process of working through the options.
Is there a page on this kind of thing at OWW? It would probably be a good example in many ways...
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