TY - JOUR
T1 - Linear Polymerization of Protein by Sterically Controlled Enzymatic Cross-Linking with a Tyrosine-Containing Peptide Loop
AU - Permana, Dani
AU - Minamihata, Kosuke
AU - Sato, Ryo
AU - Wakabayashi, Rie
AU - Goto, Masahiro
AU - Kamiya, Noriho
N1 - Funding Information:
D.P. is grateful to the Research and Innovation in Science and Technology Project (RISET-Pro) of the Ministry of Research, Technology, and Higher Education (MoRTHE) of the Republic of Indonesia (World Bank Loan No. 8245-ID) for a Ph.D. scholarship. We thank Amano Enzyme Inc. for providing us with Trametes sp. laccase. We thank James Murray, Ph.D., of Edanz Group ( www.edanzediting.com/ac ) for editing a draft of this manuscript.
Funding Information:
This study was supported by JSPS KAKENHI Grant Numbers JP16H04581, JP19H00841 (to N.K.), and JP18K14067 (to K.M.).
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/3/17
Y1 - 2020/3/17
N2 - 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, pG2pA-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 pG2pA-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-pG2pA-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.
AB - 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, pG2pA-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 pG2pA-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-pG2pA-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.
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U2 - 10.1021/acsomega.9b04163
DO - 10.1021/acsomega.9b04163
M3 - Article
AN - SCOPUS:85081674732
VL - 5
SP - 5160
EP - 5169
JO - ACS Omega
JF - ACS Omega
SN - 2470-1343
IS - 10
ER -