Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process

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.

Bacterial Inclusion Bodies: Discovering Their Better Half

“…First attempts to recover active proteins from bacterial IBs were mainly based on solubilisation of IB proteins using strong chaotropic reagents followed by subsequent renaturation procedures [37,38] (Figure 2 Apart from renaturation of IB proteins solubilised by structure-breaking chaotrops, some IB deposited proteins can be extracted in their biologically active form using mild extraction conditions (Figure 2) [40]. Although IBs are solid, mechanically stable entities, the functional fraction of IB protein appears to occur in a soluble or quasisoluble state, and it can be released in vitro by means of mild extraction protocols [55], complementing the denaturation-based strategies for IB protein recovery ( Figure 2).…”

Expression, Solubilization and Refolding Of Antigen-Binding Fragments Of Antibodies Fused With Leucine Zipper In Escherichia Coli

“…There are two important conditions to recover a high amount of bioactive proteins: first, solubilization of protein aggregates by freeze-thawing method with mild solubilization agent and reducing agent. Use of 3 M Urea preserved native-secondary structure of protein, which repress protein aggregation during refolding [5,6] and 10 mM DTT prevented incorrect disulfide bonds by reduced nonnative inter-and intramolecular disulfide bonds [7]. Second, refolding of the solubilized protein by the step-wise dialysis and the incubation with GSSG and L-arginine at the appropriate stage (1 M and 0.5 M of urea).…”

Expression and purification of human IL-2 protein from Escherichia coli

“…Additionally, the use of high incubation temperature and high IPTG concentration often leads to a high expression rate and subsequent formation of IBs (Singh et al, 2015). Therefore, attempt was made to prevent the formation of IBs by decreasing the cultivation temperature to 35 or 30°C, and by decreasing the concentration of the expression-inducing agent to 0.25 mM.…”

“…rapid transformation process, high yield of expression, and the whole expression is less expensive in comparison with other hosts (Rosano and Ceccarelli, 2014). However, the higher expression rates of recombinant proteins in E. coli are often accompanied with the formation of insoluble aggregates of the target protein called inclusion bodies (IBs) (Singh et al, 2015). The aim of this study was to express the recombinant IL-2 protein in considerable amounts that could be used as a positive control for early diagnosis of tumors and in cancer research in Egypt.…”