Abstract
Inclusion bodies are typically ignored as they are considered unwanted protein waste generated by prokaryotic host cells during recombinant protein production or harmful protein inclusions in human cell biology. However, these protein particles may have applications for in vivo immobilization in industrial biocatalysis or as cell-tolerable protein materials for the pharmaceuticals industry and clinical development. Thus, there is a need to in vivo “pull-down” (insolubilize) soluble enzymes and proteins into inclusion bodies. Accordingly, in this study, sequences from the short-chain polyphosphatase ygiF were used to design pull-down tags capable of detecting (poly)-phosphates and metal ions. These tags were compared with the entire CHAD domain from Escherichia coli ygiF and SACS2 CHAD from Saccharolobus solfataricus. The results demonstrated that highly soluble green fluorescent protein variants could be pulled down into the inclusion bodies and could have modified sensitivity to metals and di-/tri-inorganic phosphates.
Highlights
Inclusion bodies (IBs) are aggregates of partially folded or misfolded proteins
We found that the globular cellulose binding domain acted like a strong aggregation-prone module/tag. This domain was linked to the target recombinant protein using pET-34b (+) DDDK-enterokinase-SPG linker, and the 95–100% volumetric activity of the fused enzyme was effectively pulled down into active IBs (aIBs) (Nahalka and Nidetzky, 2007; Nahálka et al, 2008)
When we expressed His/Strep-tagged soluble forms of the same enzymes, despite the higher specific activities (U/g of protein) of the purified soluble proteins, aIBs were produced in the cells until the volumetric activity (U/L of fermentation broth) was the same as that of the soluble proteins, highlighting the attractiveness of aIBs as immobilized enzymes
Summary
Inclusion bodies (IBs) are aggregates of partially folded or misfolded proteins. Recent reports have shown that enzymes and proteins retain part of their biological activity when aggregated in IBs, and many biotechnological applications of IBs have been proposed, leading to introduction of the term active IBs (aIBs) (Krauss et al, 2017). AIBs have subsequently been shown to have potential applications in the production of recombinant enzymes (Jäger et al, 2020) and have emerged as a solution for in vivo enzyme immobilization in industrial biocatalysis (Ölçücü et al, 2021), as demonstrated by pull-down of Trigonopsis variabilis D-amino acid oxidase into aIBs (Nahalka and Nidetzky, 2007) and crosslinked inclusion body technology (Nahálka et al, 2008) in industrial biocatalysis. In the pharmaceuticals industry and clinical development, aIBs may have promising applications in the production of antimicrobial peptides (Köszagová and Nahálka, 2020) and nanopills (Villaverde et al, 2015).
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