(131c) Enzymatic Conjugation Strategy for the Design of Artificial Biomolecular Assemblies

Authors: 
Kamiya, N., Kyushu University
Minamihata, K., The University of Tokyo
A variety of functional biomolecular assemblies are observed in natural biological systems. In particular, proteinaceous supramolecular assemblies, such as viral capsids and bacterial surface pili, are of great interest in bioscience and bionanotechnology because their well-organized structures associated with unique biological functions would give us insight in the design of artificial biomolecular assemblies in practical applications. To mimic natural biological assembly systems, we have established site-specific protein conjugation techniques by using two enzymes with different catalytic properties.

Microbial transglutaminase (MTG) that works in crosslinking reaction between the side chains of glutamine (Gln) and lysine (Lys) residues gives us opportunity to attach an artificial affinity moiety to the selective site of protein of interest (POI). We have designed artificial substrates with multiple biotin moieties, and conducted site-specific biotinylation of POI. As a proof-of-concept study, tetrabiotinylated endoglucanase was prepared, and mixed with streptavidin. Eventually, one-dimensional assembly comprised of these protein building blocks was obtained, and it exhibited the unique hydrolytic activity for cellulosic substrates.

Another enzyme candidate is horseradish peroxidase (HRP) that catalyzes radical coupling reaction between the phenolic side chains of tyrosine (Tyr) residue. We found that HRPâ??s substrate specificity can be narrowed to a peptide tag fused genetically to POI. In line with the HRP-catalyzed polyphenol formation, we succeeded in creating proteinaceous polymeric materials via the site-specific radical coupling at a Tyr-containing tag (Y-tag). It is worth noting that both MTG and HRP provide us with site-specific and covalent conjugation strategies for a range of biomolecules, which is useful for the design of artificial biomolecular assemblies.