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タイトル: | A two-step screening to optimize the signal response of an auto-fluorescent protein-based biosensor |
著者: | Tajima, Shunsuke Nakata, Eiji https://orcid.org/0000-0001-9564-6805 (unconfirmed) Sakaguchi, Reiko Saimura, Masayuki Mori, Yasuo Morii, Takashi https://orcid.org/0000-0003-3663-3267 (unconfirmed) |
著者名の別形: | 田嶋, 竣介 中田, 栄司 才村, 正幸 森井, 孝 |
発行日: | 20-May-2022 |
出版者: | Royal Society of Chemistry (RSC) |
誌名: | RSC Advances |
巻: | 12 |
号: | 24 |
開始ページ: | 15407 |
終了ページ: | 15419 |
抄録: | Auto-fluorescent protein (AFP)-based biosensors transduce the structural change in their embedded recognition modules induced by recognition/reaction events to fluorescence signal changes of AFP. The lack of detailed structural information on the recognition module often makes it difficult to optimize AFP-based biosensors. To enhance the signal response derived from detecting the putative structural change in the nitric oxide (NO)-sensing segment of transient receptor potential canonical 5 (TRPC5) fused to enhanced green fluorescent protein (EGFP), EGFP-TRPC5, a facile two-step screening strategy, in silico first and in vitro second, was applied to variants of EGFP-TRPC5 deletion-mutated within the recognition module. In in silico screening, the structural changes of the recognition modules were evaluated as root-mean-square-deviation (RMSD) values, and 10 candidates were efficiently selected from 47 derivatives. Through in vitro screening, four mutants were identified that showed a larger change in signal response than the parent EGFP-TRPC5. One mutant in particular, 551-575, showed four times larger change upon reaction with NO and H₂O₂. Furthermore, mutant 551-575 also showed a signal response upon reaction with H₂O₂ in mammalian HEK293 cells, indicating that the mutant has the potential to be applied as a biosensor for cell measurement. Therefore, this two-step screening method effectively allows the selection of AFP-based biosensors with sufficiently enhanced signal responses for application in mammalian cells. |
著作権等: | © The Royal Society of Chemistry 2022 This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. |
URI: | http://hdl.handle.net/2433/274689 |
DOI(出版社版): | 10.1039/d2ra02226e |
PubMed ID: | 35693243 |
出現コレクション: | 学術雑誌掲載論文等 |
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