The p40 fusion domain as a scaffold for producing functional inclusion bodies.

Inclusion bodies (IBs) in Escherichia coli were once regarded as undesirable aggregates of misfolded proteins, but are now increasingly recognised as functional biomaterials. Here, we show that the N-terminal p40 domain of a lytic polysaccharide monooxygenase from Caldibacillus cellulovorans acts as a scaffold for producing functional inclusion bodies. This property was demonstrated across diverse proteins, including the fluorescent protein mCherry, three industrially relevant enzymes, and the antimicrobial peptide ZXR-2. The p40 domain consistently promoted IB formation under a wide range of induction conditions, and the resulting protein nanoparticles retained enzymatic activity, could be reused over multiple reaction cycles, and, when fused to ZXR-2, exhibited both antimicrobial and cytotoxic activities. Biophysical analyses revealed that IB size and morphology were influenced by cultivation parameters, highlighting the tunability of p40-mediated assemblies. Comparative analyses underscored the structural robustness of p40-driven IBs and their ability to support diverse protein contexts. While not intended for direct therapeutic use, these results emphasise the potential of p40 IBs as a platform technology for in vitro studies, biocatalysis, and other biotechnological applications. These findings establish the p40 fusion domain as a reliable scaffold for functional inclusion body production in E. coli, providing a foundation for future applications of IBs as versatile biotechnological tools. KEY POINTS: • p40 serves as a versatile scaffold for active protein aggregates in E. coli. • Cultivation parameters modulate aggregates accumulation and physical properties. • p40 fusions form recyclable nanobiocatalysts and stabilise antimicrobial peptides.