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

Abstract

Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/ activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the ondemand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application. Key points • Catalytically active inclusion bodies (CatIBs) are promising bionanomaterials. • Potential applications in biocatalysis, synthetic chemistry, and biotechnology. • CatIB formation represents a generic approach for enzyme immobilization. • CatIB formation efficiency depends on construct design and expression conditions.

Construction and comprehensive characterization of an EcLDCc-CatIB set—varying linkers and aggregation inducing tags

“…However studies revealed that catalytically active inclusion bodies with a reasonable residual activity can be produced by fusion of an enzyme of interest with a linker, composed of a few amino acids, and an aggregation inducing tag. Two recent reviews provided a comprehensive overview over suitable linker and aggregation inducing tags that have been successfully used for CatIB formation [ 22 , 23 ]. The aggregation inducing tags in this study are the coiled coil domain of the cell-surface protein tetrabrachion from Staphylothermus marinus (TDoT) as well as the dimeric coiled coil domain from Pseudomonas aeruginosa (3HAMP) [ 9 , 24 , 25 ].…”

New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy

“…Yet another variation on the theme of in vivo immobilisation is to use protein engineering to enable the production of catalytic inclusion bodies (CatIBs). 47 Bacterial inclusion bodies were long considered as unfolded waste material produced by heterologous over-expression of recombinant genes. Recently this opinion has changed dramatically with the application of protein engineering techniques.…”

Coiled-Coil Based Inclusion Bodies and Their Potential Applications

“…In the last years, different IB-tags, consisting of small artificial peptides or large protein domains, have been exploited to create functional IBs (Krauss et al, 2017;Jäger et al, 2020).…”

“…Typically, the tag provides the driving force for forming the intermolecular β-sheet contacts that sustain the IBs amyloid-like nanostructure. Different research groups have used coiled-coil domains as IBinducing tags (IB-tags) with significant success in the last years, suggesting that these α-helix-based tags are a feasible alternative to the β-sheet-based ones (Jäger et al, 2020).…”