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 structure-activity 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.
Highlights
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)
As the expression levels of asparaginase induced at lower temperatures (20 and 30◦C) were low, less amounts of purified inclusion bodies were produced in these cases
Inclusion bodies expressed at low temperatures were found to have lower activity in comparison to those expressed at higher temperatures
Summary
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). 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). The presence of enzyme activity, and native-like tertiary structure in inclusion bodies. 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). The aim was to study the effect of expression temperature on the quality of inclusion bodies in terms of biological activity, presence of amyloid-like structures, secondary structural content and arrangement of protein molecules inside inclusion bodies. The outcomes of the study were correlated to establish a structure-function relationship of inclusion bodies expressed at different temperatures
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