Recombinat Insoluble Protein Expression in E.coli - Inclusion body foramtion (Aug/11/2008 )

Hi..
I am new to this Forum and would like to seek your advice on my protein expression and purification.
Well, I have started expressing one of the Transcriptional related protein. It expresses pretty well (good amount of band is seen on the SDS-PAGE gel), but when I purify this using Ni-NTA loaded column I get only very less amount.
I have a suspicion that my protein is going into insoluble form and making the inclusion bodies..

How do I solve this problem and get all my protein in soluble fraction and purify it in biologically active form?

Thanks in advance.
Dam

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First of all, it really helps us if you give a bit more detail. What induction temperature / time / IPTG concentration? Do you have a gel image to show us?

There have been a few threads earlier, but here's a quick starter. Try changing the induction temperature, time and IPTG concentration. If it all still goes into the insoluble fraction (you can check this by spinning the lysate, removing the supernatant, resuspending the insoluble pellet in the same volume as the S/N, then running the samples on a gel. This gives you definite information as to the ratio of soluble and insoluble protein.

Refolding protein is quite a feasible process, so don't despair if you cannot get the protein soluble. Google 'refold database' and see what the Monash University Refold site has to say.

Good luck, and let the rest of us know your progress, especially when it works.

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