Abstract
Abstract Correct formation of disulfide bonds is crucial in the refolding of multi-disulfide-bonded inclusion body proteins. Covalent aggregation of proteins due to incorrect cross-linking of disulfide bonds decreases refolding yield. This study presents a two-step refolding method for increasing refolding yield of disulfide-bonded proteins. In this method disulfide bond formation is prevented in the first step of refolding, then in second step disulfide exchange reactions are promoted by gradual increasing of pH and cysteine concentration in refolding buffer. Recombinant tissue plasminogen activator (r-TPA, reteplase) with nine disulfide bonds was used as the model protein. Refolding yield of reteplase with two-step refolding method improved by 2.1-fold compared to dilution refolding. The successful application of two-step strategy in improving refolding yield of reteplase reveals the efficacy of this strategy for decreasing aggregation and improving correct refolding of inclusion body proteins.
Published Version
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