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Proportion of native molecules in this heterogeneous environment depends upon the conditions used during production of recombinant protein ( Ami et al., 2005 de Groot and Ventura, 2006 Villaverde et al., 2012). Structural analysis of non-classical inclusion bodies has revealed that they have less proportion of beta content suggesting less proportion of protein molecules involved in amyloid formation ( Ami et al., 2005). Non-classical inclusion bodies have been reported to be susceptible to proteolysis by non-specific proteases and are known to be easily solubilized with low concentrations of denaturants ( Upadhyay et al., 2012).
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Presence of amyloid-like structure and biological activity supports the existence of multiple protein conformations inside these aggregates. Inclusion bodies with significant biological activity are known as non-classical inclusion bodies ( Jevsevar et al., 2005 Garcia-Fruitos et al., 2012) and are characterized by loose molecular arrangement of protein molecules which can be solubilized at low concentration of denaturants like urea ( Upadhyay et al., 2012). This has opened a door to several novel biotechnological applications of inclusion body aggregates ( Roessl et al., 2010 Carvajal et al., 2011 Gatti-Lafranconi et al., 2011 Vazquez et al., 2012 Villaverde et al., 2012 Krauss et al., 2017 de Marco et al., 2019). The presence of enzyme activity, and thus, native-like tertiary structure in inclusion bodies has also been reported ( Garcia-Fruitos et al., 2005, 2007 Peternel et al., 2008 Peternel and Komel, 2010 Flores et al., 2019).
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There are several reports in support of presence of native-like secondary structures in inclusion bodies ( Oberg et al., 1994 Przybycien et al., 1994 Umetsu et al., 2004 Ventura and Villaverde, 2006).
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Inclusion bodies have been demonstrated to have amyloid like properties and are shown to contain cross-beta structures ( Carrio et al., 2005 Morell et al., 2008 Wang et al., 2008 de Groot et al., 2009 Elia et al., 2017). In contrast to the conventional view of inclusion bodies as irreversible aggregates, it has been reported that they are dynamic, reversible structures ( Carrio and Villaverde, 2001, 2002). Use of spectroscopic techniques like Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy has revealed the fine structure of inclusion body aggregates ( Wang, 2009). Formation of inclusion bodies poses a major bottleneck in high-throughput recombinant protein production and necessitates the optimization of appropriate solubilization and refolding strategies ( Singh and Panda, 2005 Singh et al., 2015a,b). High level expression of recombinant proteins in Escherichia coli often results in the formation of inclusion bodies ( Williams et al., 1982 Freedman and Wetzel, 1992 Chrunyk et al., 1993). Complex kinetics of proteinase K digestion of asparaginase inclusion bodies expressed at higher temperatures indicate higher extent of conformational heterogeneity in these aggregates. 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. Expression temperature affected the properties of asparaginase inclusion bodies. Presence of activity in inclusion bodies showed the existence of properly folded asparaginase tetramers. Purified inclusion bodies were checked for biological activities and analyzed for structural properties in order to establish a structure-activity relationship. Asparaginase was expressed as inclusion bodies at different temperatures. Here, effect of expression temperature on the quality of Escherichia coli asparaginase II (a tetrameric protein) inclusion bodies was evaluated. These aggregates have amyloid-like nature and can retain biological activity. Formation of inclusion bodies poses a major bottleneck in high-throughput recovery of recombinant protein. High level expression of recombinant proteins in bacteria often results in their aggregation into inclusion bodies.