To fold proteins containing disulfide bonds, we should consider the chemistry of the covalent bonds involved in cysteine thiol oxidation. The presence of heavy metals, and formation of mixed-disulfide intermediates. Glutathione (GSH) and oxidized glutathione (GSSG) are helpful to manage oxidative conditions, as such a GSH:GSSG ratio of 10:1 provides maximal rates of folding to the native structure for many proteins. pH also affects the condition. The rate of disulfide formation is higher in the range of pH 6 to 10 which involves the stability of folding intermediates.
Although the rate of folding is generally inverse in the concentration of denaturants, it is increased by temperature, up to that of thermal denaturation.
The rate of holding for disulfide-bonded proteins in in-vitro is generally very slow such as half-times of the order of minutes or hour, compared with its in-vivo in milliseconds.
Aggregates of unfolded proteins are actually more stable than the folded, monomeric form, resulting in formation of inclusion bodies. A major factor, which stabilizes aggregates, is hydrophobic interactions. Reducing the interaction or increasing the solubility will help to avoid the issues. A lower temperature also lessen aggregation. Nondenaturing concentration of urea or GuCl can lessen aggregation. These concentrations are made by dialysing denaturant out of the denaturation mixture. Another factor is the concentration of proteins. Definitely, a lower concentration increases folding protein and problem of recovery by “sticky” intermediates.