Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

“…Hereby, it seems reasonable to assume that conditions under which the cellular refolding and degradation machinery is outbalanced (e.g., upon conditions of strong overexpression) favor the formation of IBs. This hypothesis finds further support in recent studies, which have shown that for the same genetic construct, depending on the employed cultivation and induction conditions, either active CatIBs or classical, inactive IBs are formed (Lamm et al 2020). Here, we refer to IBs that retain a certain degree of catalytic activity (in case of enzymes) or fluorescence (in case of fluorescent reporters) as catalytically active IBs (CatIBs).…”

“…Another well-studied group of aggregation-inducing tags used for CatIB production are coiled coil domains. So far, a dimeric (3HAMP: derived from the oxygen sensor protein Aer2 of Pseudomonas aeruginosa (Airola et al 2010)) and a tetrameric coiled coil (TDoT: tetramerization domain of the cell surface protein tetrabrachion of Staphylothermus marinus (Stetefeld et al 2000)) were tested with a broad range of different target enzymes and proteins (Kloss et al 2018a;Jäger et al 2018;Diener et al 2016;Jäger et al 2019a, b;Kloss et al 2018b;Lamm et al 2020). Here, the CatIB formation efficiency was found to differ greatly depending on the target enzyme.…”

“…As for conventional IBs, temperature is the most studied cultivation parameter for CatIBs production. Generally, lower cultivation temperatures lead to CatIBs with higher activity (de Groot and Doglia et al 2008;Jevsevar et al 2005;Peternel et al 2008;Lamm et al 2020;Vera et al 2007;Wang et al 2017;Arie et al 2006), whereby lower temperature likely results in the production of a larger fraction of properly folded protein that is incorporated within the CatIB matrix. While CatIBs with higher activities can be produced at lower cultivation temperature, different studies show that lower amounts of CatIBs or less stable CatIBs are produced under those conditions (de Groot and Peternel et al 2008;Doglia et al 2008).…”

“…Due to their purity, consisting predominately of the aggregating target protein, they have traditionally been used for refolding studies, in which they served as an easy to separate source of pure target protein (Singh et al 2015). This long-held misconception has been challenged in recent years as more and more studies have revealed the dynamic, heterogeneous nature of bacterial IBs, which alongside of misfolded protein also contain protein species with amyloid structure as well as native-like and correctly folded protein (Garcia-Fruitos et al 2005;Park et al 2012;Jäger et al 2019a;Jäger et al 2018;Jäger et al 2019b;Kloss et al 2018a, b;Lamm et al 2020;Zhou et al 2012;Wang et al 2015;Jiang et al 2019;Wu et al 2011;Lin et al 2013;Diener et al 2016;Choi et al 2011;Nahalka and Nidetzky 2007;Nahalka et al 2008;Nahalka 2008;Nahalka and Patoprsty 2009;Koszagova et al 2018;Huang et al 2013;Arie et al 2006). Thus, more and more evidence suggests that those properties are to a certain degree an inherent feature of all IBs and that all cytoplasmic proteins exist in a conformational equilibrium between soluble-folded, partially misfolded, and insoluble aggregates.…”

“…Here, we refer to IBs that retain a certain degree of catalytic activity (in case of enzymes) or fluorescence (in case of fluorescent reporters) as catalytically active IBs (CatIBs). While anecdotal evidence suggests that proteins and enzymes can form CatIBs naturally (Dong et al 2014;Garcia-Fruitos et al 2005;Li et al 2013;Worrall and Goss 1989;Park et al 2012;Tokatlidis et al 1991;Krauss et al 2017;Nahálka et al 2006), the majority of studies that reported successful formation of CatIBs relied on molecular biological fusion of a variety of different aggregationinducing peptides, protein domains, or proteins (Garcia-Fruitos et al 2005;Park et al 2012;Jäger et al 2018;Jäger et al 2019a, b;Kloss et al 2018a, b;Lamm et al 2020;Zhou et al 2012;Wang et al 2015;Jiang et al 2019;Wu et al 2011;Lin et al 2013;Diener et al 2016;Choi et al 2011;Nahalka and Nidetzky 2007;Nahalka et al 2008;Nahalka 2008; Fig. 1 (Cat)IB formation in bacteria.…”