Ok, so things I have already tried include:
Different constructs (aa mutants, constructs with different N- and C-terminal tags).
Different E.coli host strains.
All resulted in inclusion bodies (even the construct with an N-terminal signal peptide that directed it to the periplasm).
Refolding has been carried out and the most important steps are described below:
1. Solubilisation of inclusion bodies with 6M GdmCl.
2. IMAC purification.
3. Reduction of cysteines with DTT.
4. Gel filtration purification - at this point the protein is approx. 99% pure).
5. Refolding by rapid dilution (although other methods have also been tried).
6. SDS-PAGE, Native-PAGE and activity assays to monitor refolding.
Unfortunately, even after extensive refolding optimisation, very low yeilds could be obtained (less than 5%).
Everything and I mean EVERYTHING was attempted in order to purify the active protein from the misfolded protein, however, it was not possible to do so.
I have almost 2 years experience in protein refolding and I can tell you that this protein just does not want to refold. The refolding problems are associated with the fact that the native protein is:
A dimer with a monomeric size of 60 kDa (dimer is 120 kDa).
Each monomer contains domains.
Each monomer contains 5 disulfide bonds.
The protein is post-translationally modified - glycosylation at 4 sites and the addition of a GPI anchor at its C-termini.
I do not wish to continue with refolding attempts and wish only to try and recover any protein with native-like secondary structure trapped within the inclusion bodies. I do not want to solubilise the inclusion bodies themselves.
I hope this has made the situation a little clearer.
Thank you for your ideas. I will now consider the possibility of using sarkosyl or triton-x. If you have any further ideas please let me know.