Catcher Domains as Multifunctional Fusion Modules for Soluble Expression, Covalent Coupling, and Spatial Organization of Recombinant Proteins

Fusion tags are essential tools for recombinant protein production, but their functions are often limited to improving solubility or facilitating purification. Catcher domains are split‐protein fragments derived from bacterial adhesion proteins that spontaneously form irreversible isopeptide bonds with their complementary Tag. Here, we describe Catcher domains as multifunctional fusion partners that not only enhance soluble protein expression but also enable modular and covalent protein assembly. Fusion of Catcher domains to diverse recombinant proteins, including both structural proteins and enzymes, remarkably enhanced their soluble expression in Escherichia coli . The covalent reaction of Catcher‐Tag pairs further provided a versatile platform for downstream modular protein engineering, allowing selective and oriented enzyme immobilization for purification and improved stability, covalent protein polymerization, and selective recruitment into compartmentalized environments. These proof‐of‐concept studies establish Catcher domains as multifunctional fusion tags that bridge soluble protein expression with modular covalent assembly and immobilization, offering a versatile strategy for the engineering of functional protein systems for applications in biotechnology.