I am trying to express and purify a bacterial membrane protein in E.coli Rosseta, after trying different conditions I've got an expression but the protein is insoluable but it seems that it doeas not form a classical inclusion body. I've tried to solubilise it with Urea and it didnt work, I've checked the cells in the light microscope and the inclusion bodies were not that evident. That made me thinking that maybe the protein is insoluable but it does not form a typical inclusion bodies and maybe it reflects a biophysical properties of the protein. Therefore I would like to purify those insoluable forms of protein without solubiliazing it. I was wondering if anyone had experiance with that kind of proteins, like fibers or sheets.
You may be able to dissolve the protein with a nondenaturing detergent, such as n-octylglucoside or Triton X-100. You can also try changing the expression conditions to favor production of soluble protein, such as by lowering the induction temperature to 16oC.
Hi Marta, do you know if your protein overexpression is killing the bugs?
Many thanks for your answers and suggestions.
I was screening the expression of the protein in different conditions: different E.coli strains, different IPTG concentration, 18, 30, and 37 degrees incubation, different growth media, different expression plasmids. In some conditions there was a fraction of the protein which was soluable (by soluable/insoluable I mean present in the supertnatant/pellet after the cell lysis and low speed centrifugation). At first sight the protein behaved like an inclusion body, but was not dissolving in Triton-X nor 8M urea. Probably I could further screen for solubilisation conditions. But I want to try to purify the non-solubilized protein, so some how wash it and recover it from the pellet in the insoluable form as it is. I was wondering if anyone has experiance on that?
Inclusion body proteins are often a mess of misfolded and denatured proteins. If your protein has a simple structure, then using denaturants and/or detergents could give a good yield.
If your protein is conformationally sensitive, then I would recommend to harvest the bugs before they deposit it into inclusion bodies.
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