In some papers people have recommended to use incubation at 42 degree celsius for short time for protein expression after IPTG induction? If YES, What will be its effect on protein's solubility?
Note: It is Feasible to use 42 degree celsius with E.coli expression systems.
I would recommend the opposite: grow the cells at 37C, when they reach mid-log (OD600~0.6) move them to 18C (eighteen) for ~1h and then add inducer for 5~6 hours to full O.N. The logic is that slower expression will avoid aggregation.
Another trick I have seen is growing the cells in the presence of high NaCl in the medium (I think final wad 1M). The cells accumulate osmolants that should facilitate proper folding.
I did not heard about expression at 42C but would imagine it is based on induction of chaperons. If this is indeed the case I would suggest a 30 min 42C heat shock, adding inducer and returning the cells to 37C. I think over-expression in 42C is likely to result miss-folded proteins.
Yoram
how long is this short time? If this short time is few minute, maybe it's for starting expression. when temperature increased, the time of expression decreased for achieving the enough amount of protein that you need. but as you said higher temperatures cause aggregation of desired protein ( even is themoprotein)
It depends on the protein and the E. coli in consideration. In case of preparation of lambda phage, it is common to do the initial infection and early propagation at lower temp but induction and lysis of cell is conducted at ~ 40C.
Not every E. coli strain is too happy at 42C but some do quite well, to the point that post IPTG induction they can produce so much protein that the level can reach 24-30% of total cellular protein content. The time has to be optimized for each combination to get optimum yield, too short a time will not be enough for sufficient accumulation of protein and too much time will cause cell lysis - test around 2-4hr range at time points 30 min apart.
@younesi and @tousif sir.. thanx for your response. In my case I have got significant expression in one of my thermophilic protein in rossetta DE3 at 42C after 2 hrs of post induction. But again solubility is a problem.
I would recommend the opposite: grow the cells at 37C, when they reach mid-log (OD600~0.6) move them to 18C (eighteen) for ~1h and then add inducer for 5~6 hours to full O.N. The logic is that slower expression will avoid aggregation.
Another trick I have seen is growing the cells in the presence of high NaCl in the medium (I think final wad 1M). The cells accumulate osmolants that should facilitate proper folding.
I did not heard about expression at 42C but would imagine it is based on induction of chaperons. If this is indeed the case I would suggest a 30 min 42C heat shock, adding inducer and returning the cells to 37C. I think over-expression in 42C is likely to result miss-folded proteins.
Yoram
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology