Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

Abstract

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.

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

“…These approaches, however, have their limitations in terms of insufficient control over enzyme stoichiometry or sensitivity to environmental conditions (e.g., pH and ion-strength of reaction media), respectively. In contrast, our method is based on robust covalent linkage that is controlled by the versatile SpyTag/SpyCatcher crosslinking technology and even allows for programming of stoichiometries other than the equimolar ratio shown here 29 .…”

“…Hydrogels are porous polymers that can be constructed from natural or synthetic structural proteins 23,24 . A recently established protein gelation strategy utilizes a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions [25][26][27][28][29] . While these protein hydrogels are being explored for applications in biomedical sciences, such as cell encapsulation and tissue engineering, strategies for their exploitation in biocatalysis remain underdeveloped.…”

Post‐translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase

“…Substrate channelling is an effective strategy for creating synthetic enzyme pathways, and using protein scaffolds to assemble enzymes with increased catalytic activity can be particularly useful in coupling enzymes that work synergistically. A polymer of SpyCatcher was recently applied to fabricate protein assemblies by using SpyTagged enzymes (Figure A) . This construct containing SpyCatcher and horseradish peroxidase (HRP) as a catalyst allowed a synthetic scaffold to be manufactured for the modular assembly of various tagged enzyme complexes, arabinofuranosidase, and endoxylanase in a ratio‐controlled manner.…”

“…Additionally, Jia, et al. used SpyTag/SpyCatcher system to generate a biosensing module, capable of detecting the model antigen, ovalbumin . SpyCatcher polymer was employed as a scaffold to conjugate SpyTagged Nanoluc luciferase and SpyTagged Protein G, then subsequently used as a bioprobe for ELISA, allowing ovalbumin to be detected.…”

“…Substrate channelling is an effective strategy for creating synthetic www.chembiochem.org enzyme pathways, and using protein scaffolds to assemble enzymesw ith increased catalytic activity can be particularly useful in coupling enzymes that work synergistically.Apolymer of SpyCatcher was recently appliedt of abricate protein assemblies by using SpyTagged enzymes ( Figure 2A). [34] This construct containingS pyCatcher and horseradish peroxidase (HRP) as ac atalyst allowed as ynthetic scaffoldt ob em anufactured for the modular assembly of various tagged enzyme complexes, arabinofuranosidase, and endoxylanase in ar atio-controlled manner.T his assembly achieved higher conversion of sugars than free enzymes due to substrate channelling from the spatial proximity of the co-operative enzymes.A dditionally, Jia, et al used SpyTag/SpyCatcher system to generate ab iosensing module, capable of detecting the model antigen, ovalbumin. [34] SpyCatcher polymer was employed as as caffold to conjugate SpyTagged Nanolucl uciferase and SpyTagged Protein G, then subsequently used as ab ioprobe for ELISA,a llowing ovalbumin to be detected.…”