Inhibition of aggregation side reactions during in vitro protein folding.

Publisher Summary When synthetic or natural genes or cDNAs are overexpressed in the cytosol of microbial host cells such as Escherichia coli, recombinant proteins can be produced in large amounts. Although high-level expression is usually achieved using standard recombinant DNA techniques, the polypeptides are often sequestered in the form of insoluble, inactive inclusion bodies. These large, dense particles often span the whole diameter of the host cell. After proper isolation they consist primarily of the recombinant protein. Active protein can be recovered from the inclusion bodies by solubilization in chaotrophic buffer systems and subsequent in vitro folding. However, unproductive side reactions (predominantly aggregation) often compete with correct folding during in vitro folding. Various techniques, some of which are summarized in this chapter, have been developed to inhibit aggregation side reactions and to ensure efficient in vitro protein folding. These methods, which can now be considered as standard laboratory techniques, allow the refolding of many inclusion body proteins on the laboratory scale or even in industrial production processes.