Investigating the SpyCatcher-SpyTag system of making multimodular proteins

Abstract

Cellulose is commonly used as the stiff component to interact with a softer matrix, such as proteins, to form composites with excellent mechanical properties. Carbohydrate-binding modules (CBMs) are a very diverse type of proteins that can specifically interact with cellulose. Research on double CBMs proteins associating with cellulose nanofibrils (CNFs) suggests that proteins modules with an interlinking architecture can be used to tune the mechanical properties of natural materials. However, there is still a great need for studying the role of multiple CBMs in composites and describing the underlying mechanic principles in a unified manner. Therefore, engineering multi-CBM proteins are great of importance both for understanding and manipulating CBM proteins in composite materials.

By making use of this recently developed SpyCatcher-SpyTag system, we successfully engineered multi-CBM proteins and investigated effects that could have an impact on the multi-CBM proteins formation. SpyTag is a peptide of 13 amino acids that specifically forms a covalent isopeptide bond with its protein partner SpyCatcher. This system has been extensively used in different applications, such as hydrogels, labelling, vaccines generation, and protein assembly. Here, SpyCatcher was genetically fused with one or two CBMs and ligated by different bivalent SpyTag proteins to form multi-CBM proteins. The best yield of multi-CBM proteins was 60% by mixing SpyCatcher fused to two CBM domains with two SpyTag linked by a linker of 22 amino acids. The reaction can be implemented under a wide range of condition with temperature from 4 to 37°C, pH from 5 to 8 and salt concentration lower than 200 mM. The accessibility of SpyCatcher reaction pocket and the complete structure of SpyTag are key points to produce multi-CBM proteins with a good performance.

Using SpyCatcher-SpyTag system to construct multi-CBM proteins is a robust and flexible way and can be used to investigate the functional effect of CBMs in cellulose nanocomposites. Furthermore, it opens a gate to construct proteins with multimodular in an easy way.