As has been suggested, you can try other hosts - for instance Gram positive bacteria (Bacillus megaterium is an interesting host as are other bacilli, especially if your protein is secreted), other Gram negative bacteria (Pseudomonas for instance),
Can anyone comment on availability of Archaea expression systems? Based on available structures, it would appear that their chaperons are much more similar to eukaryotic one than anything present in eubacteria. For a long time, I've been puzzling over why there is still no popular/workable Archaea expression system. Reason?
yeasts (Pichia, Hansenula, Yarrowia, Klyveromyces), insect cells (Trichoplusia or Spodoptera - they're different!)
I've compared Sf9/Sf21 and High5 cells for many, many baculoviruses and have yet to find a difference that would ultimately matter. One clear difference is that Trichoplusia/High5 seem to require less virus to get to the same level of expression - but that level is exactly the same as what Sf9 give at higher pfu/cell infections. So the benefit is only felt by people who failed to amplify their virus properly. Same with regard to solubility. Some of the proteins I've dealt with misfold (partially or completely) even in insect cells but back to back comparison of High5 and Sf9 showed no difference. Trichoplusia are a lot more difficult to get growing in suspensions without horrible clumping.
mammalian cells (dozens of options there too)
All of them are essentially equivalent = pathetic, as far as overexpression is concerned. One exception though: transient expression in Cos-1 cells. It's not a system one can do on a large scale but the amount of protein/cell is 10-20X higher than constitutive expression and thus it can be appropriate to to get enough material for limited proteolysis or biochemical assays that don't require lots of protein.
, or cell-free expression. If you're truly desperate you can try expression in more esoteric hosts such as plant roots, algi, parasites (Leishmania), whole insect larvae, animals (milk), and so forth.
Dictyostelium discoideum (slime mold) deserves honorary mention here. Its cultures are nearly as cheap as E.coli, it grows almost as fast as yeasts, it doies not have funky post-translational processing yeasts frequently do, and it gives at least as much protein as common mammalian expression systems. The drawback is its weird codon usage but with the gene synthesis prices coming down fast it's fast becoming a viable option.
I got enclusion body in many cases when I tried to express human proteins in E coli. I would like some suggestions on how to go about it. I would also like to try co-expression of GroEL/GroES or DnaJ/DnaK, and would like to know where to get the plasmids.
IMHO, if you don't get *any* soluble expression at all in minimal medium expressing at 16C, the chances that anything you do in E.coli will magically make soluble protein are slim to none. If, however, there is *some* folding but it inefficient, then, As Artem mentioned, there is a long list of options you can consider... Good luck, Dima
