Hi Michael, It worked for me with one viral enzyme but did not work with 2 others. In the successful case, I used Takara's chaperone plasmids to screen for the best composition of chaperones. The GroEL-GroES improved the yield of the soluble enzyme, but I got same activity (per 1L of media) as before. The plasmid with chaperone tig gave the biggest increase in activity (~4-8 fold). Still, the yield was rather low, in a range of 1 mg of purified protein per 1L of media. I also had to optimize the expression time and temperature to get best results. They were ~14 h at 17-20C. Interestingly, the protein failed to express in insect cells (was too poisonous for them), so the combination of a solubilizing domain, chaperones and Ecoli expression were the only available option for me.
Regards Alex On Apr 22, 2011, at 8:28 AM, Kothe, Michael wrote: > Dear ccp4bb, > > I am curious to hear of examples where the expression of well-behaved protein > was achieved by the coexpression of chaperones in E. coli. I know the > appropriate strains and vectors exist, but I can’t remember hearing of a > successful case. I have heard anecdotally of several cases where it was tried > without success (including one attempt I made myself). I also heard of > concerns that the chaperones might copurify with the (now soluble) protein of > interest and are difficult to remove. > > Thanks, > Michael >
