Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post‐translational modification methods is particularly favored since the proteins can be site‐specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher–SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio‐controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA. Co‐localizing biomolecules on synthetic scaffolds has been successfully develop to achieve better performance than single molecules in many applications. In this study, a polymeric SpyCatcher scaffold for varieties of biological uses is prepared through catalytic reaction of horseradish peroxidase. The results show it possesses the applicability to site‐specifically conjugate different biomolecules at arbitrary ratio. Our results indicate that the developed SpyCatcher polymer is a robust and versatile protein scaffold that is suitable for multiple uses in a range of applications in bioindustry.