Structure-Function Relationship of Inclusion Bodies of a Multimeric Protein

Abstract

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.

Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

“…Amyloid-like properties were seen in β-galactosidase IBs expressed in E. coli , which were shown to be biologically active ( Carrió et al, 2005 ). Another study carried out by Singh et al (2020) to characterize asparaginase IBs confirmed the high β-content as well as the catalytic activity of the amyloid-like aggregates. Furthermore, it was determined that temperature could modulate the levels of reported amyloid features ( Singh et al, 2020 ).…”

“…Another study carried out by Singh et al (2020) to characterize asparaginase IBs confirmed the high β-content as well as the catalytic activity of the amyloid-like aggregates. Furthermore, it was determined that temperature could modulate the levels of reported amyloid features ( Singh et al, 2020 ). The parallel findings were reported in a similar study ( Žerovnik et al, 2007 ).…”

“…Amorphous aggregation is a thermodynamically favorable process obviating the need for a high energy barrier necessary for nucleation processes such as amyloid fibrillation and crystallization ( Yoshimura et al, 2012 ). The catalytic activity were found for some amyloid aggregates which are characterized by cross β-sheet motifs ( Chapman et al, 2002 ; Carrió et al, 2005 ; Jordal et al, 2009 ; Upadhyay et al, 2012 ; Singh et al, 2020 ). Conversely, E. coli protein IBs appear as amorphous aggregates lacking proper structure and function.…”

“…Conversely, E. coli protein IBs appear as amorphous aggregates lacking proper structure and function. Despite this, a growing body of evidence has suggested that certain IBs proteins may possess amyloid-like structures with an associated functionality ( Carrió et al, 2005 ; Upadhyay et al, 2012 ; Singh et al, 2020 ). Amyloid-like properties were seen in β-galactosidase IBs expressed in E. coli , which were shown to be biologically active ( Carrió et al, 2005 ).…”

“…Second, the propensity of IBs to aggregate homogenously with a high density presents an opportunity for quick isolation of expressed proteins ( Ramón et al, 2014 ). Moreover and in certain cases, research has demonstrated that inclusion bodies formed by certain proteins might contain bio-functional preparations ( Singh et al, 2020 ; Singhvi et al, 2020 ) despite the earlier common notion of mostly un-functional and inactive protein complexes within purified IBs. Such promising observations can be further investigated for the factors that affect such functionalities and decipher the mechanism(s) behind the observed differences among individual proteins.…”