Cytoplasmic expression of recombinant proteins requiring disulfide bridges in Escherichia coli usually leads to the formation of insoluble inclusion bodies (IBs). The reason for this phenomenon is found in the reducing environment of the cytoplasm, preventing the formation of disulfide bridges and therefore resulting in inactive protein aggregates. However, IBs can be refolded in vitro to obtain the protein in its active conformation. In order to correctly form the required disulfide bridges, cystines are fully reduced during solubilization and, with the help of an oxidizing agent, the native disulfide bridges are formed during the refolding step. Here, a protocol to identify suitable redox conditions for solubilization and refolding is presented. For this purpose, a multivariate approach spanning the unit operations solubilization and refolding is used.
Keywords: Design of Experiment (DoE); Disulfide bridges; Inclusion bodies (IBs); Inclusion body refolding; Redox system; Unit operation-spanning.
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