Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

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High-level expression of recombinant proteins in Escherichia coli often results in accumulation of protein molecules into aggregates known as inclusion bodies (IBs). Isolation of properly folded, bioactive protein from IBs is a cumbersome task and most of the times results in poor recovery. The process of recovering bioactive proteins from IBs consists of solubilization of IB aggregates using denaturants, followed by refolding of the solubilized protein. Here, we describe a simple protocol for screening of buffers for solubilization of IB proteins. Various IB aggregate solubilization methods including organic solvents have been described.

High level expression of recombinant proteins in bacteria often results in their aggregation into inclusion bodies. Formation of inclusion bodies poses a major bottleneck in high-throughput recovery of recombinant protein. These aggregates have amyloid-like nature and can retain biological activity. Here, effect of expression temperature on the quality of Escherichia coli asparaginase II (a tetrameric protein) inclusion bodies was evaluated. Asparaginase was expressed as inclusion bodies at different temperatures. Purified inclusion bodies were checked for biological activities and analyzed for structural properties in order to establish a structureactivity relationship. Presence of activity in inclusion bodies showed the existence of properly folded asparaginase tetramers. Expression temperature affected the properties of asparaginase inclusion bodies. Inclusion bodies expressed at higher temperatures were characterized by higher biological activity and less amyloid content as evident by Thioflavin T binding and Fourier Transform Infrared (FTIR) spectroscopy. Complex kinetics of proteinase K digestion of asparaginase inclusion bodies expressed at higher temperatures indicate higher extent of conformational heterogeneity in these aggregates.