Rational truncation of the p40 aggregation-inducing domain enables tailored formation of functional bacterial inclusion bodies for biocatalysis

Recombinantly produced bacterial inclusion bodies (IBs) have emerged as versatile biomaterials for biocatalysis, enzyme immobilisation, and process-relevant biotechnological applications. Fusion of aggregation-inducing protein domains enables the formation of functional IBs; however, identifying optimal fusion architectures typically relies on empirical screening. Here, we present a rational strategy to derive shorter, designable aggregation-inducing variants from the p40 domain, a previously established IB-forming scaffold. Sequence- and structure-based aggregation predictions were used to identify aggregation-prone regions within p40, guiding the design of six truncated variants containing defined aggregation hotspots. These variants were fused to four target proteins of increasing structural complexity and systematically evaluated for IB formation, morphology, protein content, and retained functionality. We show that p40 truncation length strongly influences aggregation behaviour and functional incorporation in a target-dependent manner, enabling the tuning of IB properties without loss of reversible aggregation capability. Notably, selected p40 truncation variants preserved compatibility with reversible aggregation and post-assembly material functionalisation, supporting their use as refined aggregation-inducing fusion partners. This work positions p40 truncation as a molecular engineering framework enabling the controllable design of functional IBs for downstream bioprocess and biocatalytic applications. • Rational p40 truncation enables tailored formation of functional inclusion bodies • Aggregation behaviour depends on p40 length and target protein context • Truncated p40 variants enhance active enzyme loading in CatIBs • CatIB size, morphology and kinetics can be modulated via fusion construct design • p40 truncations preserve reversible aggregation and material functionalisation