Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications

State-of-the-art and novel approaches to mild solubilization of inclusion bodies

“…While initially inclusion bodies (IBs) were thought to be aggregates of misfolded protein ( García-Fruitós et al, 2012 ), they have been shown to contain correctly folded secondary and tertiary protein structures, in some cases even with biological activity ( García-Fruitós, 2010 ; Villaverde et al, 2015 ; Belkova et al, 2022 ; López-Cano et al, 2022 ). Preserving these existing structures during the solubilization step has been correlated with an increase in refolding yield due to reduced re-aggregation ( Singh et al, 2015 ; Kachhawaha et al, 2022 ). Thus, the concept of “mild solubilization” originated, describing the solubilization of IBs without fully denaturing the protein.…”

Inclusion Bodies in Ionic Liquids

“…This recognition broadened the scope of research on inclusion bodies and led to novel strategies for refolding processes, their roles as protein storage mediums, and their direct applications in nanotechnology and biotechnology. In the context of inclusion body processing, the workflow can be broadly categorized into two main segments: upstream and downstream processing [4,5]. The upstream segment encompasses microbial fermentation [6] and subsequent cellular harvesting.…”

“…The next stage of the downstream processing is refolding (green sphere in Figure 1), wherein the target protein undergoes a transition from a (partially) unfolded state to its native functional conformation [4,10]. Initiation of the folding reaction is primarily achieved by removing chaotropic agents by diluting the solution with an appropriate refolding buffer [11].…”

“…Inclusion bodies represent heterogeneous entities characterized by significant variations in their morphology, architecture, organizational features, and functional roles [4]. Given this inherent complexity, a comprehensive array of analytical techniques is frequently employed to decipher their intricate structural and functional fingerprints.…”

“…Given this inherent complexity, a comprehensive array of analytical techniques is frequently employed to decipher their intricate structural and functional fingerprints. As several experimental techniques to promote the theoretical understanding of inclusion bodies have been reviewed by others [4,7], we briefly mention the most important methods here.…”