Development of bioluminescent Switchbody, antigen-triggered enzyme switch and elucidation of its principle
DOI:
https://doi.org/10.51094/jxiv.1096Keywords:
protein switch, homogeneous immunoassay, X-ray structural analysis, solution NMR, MD simulationAbstract
We developed an enzyme switch, Switchbody, by integrating an antibody with a fragment of a split enzyme for precise enzyme activity regulation in response to an antigen. Using NanoLuc luciferase as the split enzyme, we engineered a luciferase-based Switchbody by fusing its fragment, HiBiT, to the N-terminus of antibody, and the Switchbody detected antigens in a dose-dependent manner with the complementary fragment, LgBiT, and its substrate. ELISA showed that interaction between HiBiT and LgBiT was enhanced by antigen binding. Moreover, X-ray crystallography and NMR revealed the heterogeneous trapped state of the HiBiT region and increased motility upon antigen binding, respectively. MD simulations and luminescence measurements showed that antigen disrupted the trapping of HiBiT in the antibody, enabling its release. By applying this "Trap & Release" principle to Protein M, an antibody binding protein, we successfully converted label-free IgG antibodies into bioluminescent immunosensors, demonstrating its versatility. The principle in Switchbody has the potential to expand switching technology beyond luciferase to other various enzymes in the future.
Conflicts of Interest Disclosure
T.Y., B.Z., H.U., and T.K. received honoraria from HikariQ Health, Inc. for another unrelated project.Downloads *Displays the aggregated results up to the previous day.
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Takanobu Yasuda
Yoshiyuki Ueno
Masahiko Taguchi
Naoya Tochio
Hiromasa Yagi
Shuma Yazaki
Ryoichi Arai
Bo Zhu
Takanori Kigawa
Hiroshi Ueda
Tetsuya Kitaguchi
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