No abstract

The polymerization of proteins can create newly active and large biomacromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N-and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O 2 to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H 2 O 2 . The addition of H 2 O 2 in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.

The structure of a protein complex needs to be controlled appropriately to maximize its functions. Herein, we report the linear polymerization of bacterial alkaline phosphatase (BAP) through the site-specific cross-linking reaction catalyzed by Trametes sp. laccase (TL). We introduced a peptide loop containing a tyrosine (Y-Loop) to BAP, and the Y-Looped BAP was treated with TL. The Y-Looped BAP formed linear polymers, whereas BAP fused with a C-terminal peptide containing a tyrosine (Y-tag) showed an irregular shape after TL treatment. The sterically confined structure of the Y-Loop could be responsible for the formation of linear BAP polymers. TL-catalyzed copolymerization of Y-Looped BAP and a Y-tagged chimeric antibody-binding protein, pG 2 pA-Y, resulted in the formation of linear bifunctional protein copolymers that could be employed as protein probes in an enzyme-linked immunosorbent assay (ELISA). Copolymers comprising Y-Looped BAP and pG 2 pA-Y at a molar ratio of 100:1 exhibited the highest signal in the ELISA with 26-and 20-fold higher than a genetically fused chimeric protein, BAP-pG 2 pA-Y, and its polymeric form, respectively. This result revealed that the morphology of the copolymers was the most critical feature to improve the functionality of the protein polymers as detection probes, not only for immunoassays but also for other diagnostic applications.