I've been trying to solubly express my protein in e. coli for the last year and a half. I have created fragments and tried them, and those themselves have problems. My protein is about a 1000 amino acids (112kDa) and always goes to the pellet no matter what I do. I'm pretty sure it's going into the inclusion bodies, as I have tried membrane preps with a few different detergents and it still goes into the pellet. I also fused my protein to MBP, but after cleavage my protein fell out of solution.
Recently I resuspended my pellet in 6M urea and was able to extract my protein from the pellet, which got me thinking of trying to refold my protein. I know this is also a long arduous process, and before I attempt it my biggest fear is what if I get a incorrectly folded protein? I don't want to do a bunch of experiments only to find out it was misfolded. This scares me because to date, no one has purified this protein so I have no way of knowing if it is correctly folded. I would follow up the refolded protein my CD, and maybe crystallography.
Thanks for any advice.