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Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells
N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical pro...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8200523/ https://www.ncbi.nlm.nih.gov/pubmed/34136513 http://dx.doi.org/10.3389/fnut.2021.621273 |
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author | Naz, Ruphi Okla, Mohammad K. Fatima, Urooj Mohsin, Mohd. Soufan, Walid H. Alaraidh, Ibrahim A. Abdel-Maksoud, Mostafa A. Ahmad, Altaf |
author_facet | Naz, Ruphi Okla, Mohammad K. Fatima, Urooj Mohsin, Mohd. Soufan, Walid H. Alaraidh, Ibrahim A. Abdel-Maksoud, Mostafa A. Ahmad, Altaf |
author_sort | Naz, Ruphi |
collection | PubMed |
description | N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels. |
format | Online Article Text |
id | pubmed-8200523 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-82005232021-06-15 Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells Naz, Ruphi Okla, Mohammad K. Fatima, Urooj Mohsin, Mohd. Soufan, Walid H. Alaraidh, Ibrahim A. Abdel-Maksoud, Mostafa A. Ahmad, Altaf Front Nutr Nutrition N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels. Frontiers Media S.A. 2021-05-31 /pmc/articles/PMC8200523/ /pubmed/34136513 http://dx.doi.org/10.3389/fnut.2021.621273 Text en Copyright © 2021 Naz, Okla, Fatima, Mohsin, Soufan, Alaraidh, Abdel-Maksoud and Ahmad. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Nutrition Naz, Ruphi Okla, Mohammad K. Fatima, Urooj Mohsin, Mohd. Soufan, Walid H. Alaraidh, Ibrahim A. Abdel-Maksoud, Mostafa A. Ahmad, Altaf Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title | Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title_full | Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title_fullStr | Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title_full_unstemmed | Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title_short | Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells |
title_sort | designing and development of fret-based nanosensor for real time analysis of n-acetyl-5-neuraminic acid in living cells |
topic | Nutrition |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8200523/ https://www.ncbi.nlm.nih.gov/pubmed/34136513 http://dx.doi.org/10.3389/fnut.2021.621273 |
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