Hello,
am I right that in Inclusion body processes the chemicals mentioned above are mainly used for cell disruption and removal of cell debris? Which of them do I use for solubilization of the IBs?
Do they have any role in the refolding of the IBs?
Triton X-100 and lysozyme are added to lyse the cells; DNase decreases the viscosity of the extract and EDTA is optional. None of these will solubilize inclusion bodies. For this, you will need a chaotropic agent such as urea > 5 M or guanidine hydrochloride >3 M. Refolding requires removal of the chaotrope by dilution or dialysis, but finding proper conditions for efficient refolding is usually tricky, since most of the time, dialysis of the chaotrope leads to precipitation of the misfolded protein. In most cases, it is of advantage to optimize production conditions such that the target protein is produced in soluble form.
The EDTA is added to inhibit metallo-proteinases, which would/could chop up your protein. In addition, it protects somewhat the cysteine thiol-groups. The sulfur atom likes to interact with metal ions.
Thans for your helpful answers. Perhaps you might want to comment on my other question:
https://www.researchgate.net/post/What_is_a_common_ph-Level_for_cell_broth_when_harvesting_IgG_antibodies_in_a_cell_bank_and_at_a_first_centrifugation_and_filtration_step_afterwards
Hi Dennis,
I do not recommend to refold proteins, unless it is indispensable. Have you tried to express the protein using sorbitol and betaine? sometimes this can improve the solubilization of your protein. Anyway, you can use a redox system in your refolding buffer, this also can help the refolding if your protein has disulfide bonds.
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