A two-step screening to optimize the signal response of an auto-fluorescent protein-based biosensor. Issue 24 (20th May 2022)

• Record Type: Journal Article
• Authors: Tajima, Shunsuke; Nakata, Eiji; Sakaguchi, Reiko; Saimura, Masayuki; Mori, Yasuo; Morii, Takashi
• Is Part Of: RSC advances. Volume 12:Issue 24(2022)
• Page Start: 15407
• Page End: 15419
• Publication Date: 2022-05-20
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2ra02226e
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : A two-step screening procedure allows optimization of the optical response of an auto-fluorescent protein-based biosensor for nitric oxide without structural information. Abstract : 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 H2 O2 . Furthermore, mutant 551-575 also showed a signal response upon reaction with H2 O2 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.