I'm trying to refold the cysteine-rich bovine pancreatic trypsin inhibitor (BPTI) with a glutathione(reduced) / glutathione(oxidized) - redox system. I use a Tris-buffer at pH 8.
First I reduced native BPTI with TCEP to obtain the unfolded peptide/protein chain. Afterwards I desalted the solution by gel filtration.
Now several attempts to refold the reduced chain to its native form have not been successful. I followed the procedings given in multiple papers.
I always seem to have so precipitate in my folding solution.
The precipitate is soluble at strong basic conditions, however it has a much higher retention time in HPLC than the native BPTI.
Has anyone experience with this problem?
Under described conditions I suppose that large interprotein aggregates may be formed due trans-sulfuration exchage reactions. I mean that interproten SS bridges are formed. You can verify this with an SDS electrophoresis under non reducing conditions. Also by adding DTT to your precipitate you should dissolve it. Strong basic conditions break disulfide bridges similarly to DTT. You could try different pHs and different GSH/GSSG ratios in order to obtain better results.
Ranieri Rossi thank you for your answer and your suggestions. I'll try to find better folding conditions (new buffer, different pH, other ratios of GSH/GSSG).