posted on 2021-07-12, 19:14authored byKosuke Minamihata, Yusuke Tanaka, Pugoh Santoso, Masahiro Goto, Dan Kozome, Toki Taira, Noriho Kamiya
Enzymatic
reaction offers site-specific conjugation of protein
units to form protein conjugates or protein polymers with intrinsic
functions. Herein, we report horseradish peroxidase (HRP)- and microbial
transglutaminase (MTG)-catalyzed orthogonal conjugation reactions
to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD,
and chitin-binding domain, LysM2. We engineered the ChiA
and CatD by introducing a peptide tag containing tyrosine (Y-tag)
at N-termini and a peptide tag containing lysine and tyrosine (KY-tag)
at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM2 with Y-tag and KY-tag (Y-LysM2-KY) or with a glutamine-containing
peptide tag (Q-tag) (LysM2-Q) were constructed. The proteins
with Y-tag and KY-tag were efficiently polymerized by HRP reaction
through the formation of dityrosine bonds at the tyrosine residues
in the peptide tags. The Y-CatD-KY polymer was further treated by
MTG to orthogonally graft LysM2-Q to the KY-tag via isopeptide
formation between the side chains of the glutamine and lysine residues
in the peptide tags to form LysM2-grafted CatD polymer.
The LysM2-grafted CatD polymer exhibited significantly
higher antifungal activity than the homopolymer of Y-ChiA-KY and the
random copolymer of Y-CatD-KY and Y-LysM2-KY, demonstrating
that the structural differences of artificial chitinase polymers have
a significant impact on the antifungal activity. This strategy of
polymerization and grafting reaction of protein can contribute to
the further research and development of functional protein polymers
for specific applications in various fields in biotechnology.