Abstract
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.
Highlights
High level expression of recombinant protein in Escherichia coli often results in aggregation of the expressed protein molecules into inclusion bodies [1,2,3]
It was observed that same type of protein molecules co-aggregate in cell whereas different proteins like VP1 do not co-aggregate with amyloid beta [25]. These results showed that protein aggregation in inclusion bodies is a highly specific phenomenon
Advanced structural techniques have significantly enhanced our understanding of protein structure in inclusion body aggregates
Summary
High level expression of recombinant protein in Escherichia coli often results in aggregation of the expressed protein molecules into inclusion bodies [1,2,3]. Active protein molecules can be extracted from non-classical inclusion bodies using mild solubilization conditions This process preserve the native-like protein structures present in inclusion bodies and bypasses the refolding steps. Low concentration of urea in many cases has been used to solubilize inclusion body aggregates [46,63] These solubilization agents result into extraction of active recombinant protein without any requirement of refolding step. Use of organic solvents like β-mercaptoethanol (βME) [70] and n-propanol in combination with low concentration of urea has been reported as a novel solubilization strategy for improved recovery of proteins [60] Both the solvents have been used to solubilize human growth hormone inclusion bodies. Common additives used in refolding buffer include chaotropic agents like urea [19] or guanidine hydrochloride [74] in low concentrations, amino acids like glycine [100], arginine [101,102] and proline
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