SavedAdd your journal

Journals / Sakarya University Journal of Science / 2021 / Cilt: 25 Sayı: 5

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

İbrahim İNCİR
Özlem KAPLAN
Sema BİLGİN
İsa GÖKÇE
Pages
1235–1244
Publication Date
2021-10-30
DOI
—
🏛️Bibliographic metadata and full text access for this article are provided via DergiPark.
DergiPark Record ↗
✨

AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:Proteases are closely associated with many pathological conditions.
📊
Key Findings & Data:The plasmid encoding the mNeonGreen-mRuby3 fluorescent protein-based FRET biosensor was constructed.
💡
Conclusion & Impact:The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.
🔬Design:Modeling & Simulation
📍Scope:Comprehensive Sample
⚙️Variables:Recombinant DNA Technology · Fluorescent Proteins · Fluorescent Resonance Energy Transfer (FRET)

Abstract

Proteases are closely associated with many pathological conditions. Efficient detection of protease activity may be useful for diagnosis, prognosis, and the development of new therapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as the non-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins are widely used in FRET biosensors because they can be genetically encoded and compatible with cells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. In this study, it was aimed to detect protease activity using an FP-FRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding the mNeonGreen-mRuby3 fluorescent protein-based FRET biosensor was constructed. The gene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells using recombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed in the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.

🔖

Cite This Article

İNCİR, İ., KAPLAN, Ö., BİLGİN, S., GÖKÇE, İ. (2021). Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity. Sakarya University Journal of Science, Cilt: 25 Sayı: 5, 1235–1244. https://ajindex.com/articles/941766

📄 Full Text PDF Viewer

📄

PDF görüntüleyici hazırlanıyor...

References (13)

  1. J. S. Bond, “Proteases: History, discovery, and roles in health and disease,” Journal of Biological Chemistry, vol. 294, no. 5, pp. 1643–1651, 2019, doi: 10.1074/jbc.TM118.004156
  2. B. T. Bajar, E. S. Wang, S. Zhang, M. Z. Lin, and J. Chu, “A guide to fluorescent protein FRET pairs,” Sensors (Switzerland), vol. 16, no. 9, 2016, doi: 10.3390/s16091488
  3. M. P. Maria-Chantal Chirio-Lebrun, “Fluorescence resonance energy transfer (FRET): theory and experiments,” Biochemical Education, vol. 26, no. 4, pp. 320–323, 1998
  4. A. Kaur, P. Kaur, and S. Ahuja, “Förster resonance energy transfer (FRET) and applications thereof,” Analytical Methods, vol. 12, no
  5. Royal Society of Chemistry, pp. 5532–5550, Dec. 14, 2020. doi: 10.1039/d0ay01961e
  6. A. Masharina, L. Reymond, D. Maurel, K. Umezawa, and K. Johnsson, “A Fluorescent Sensor for GABA and Synthetic GABA,” Journal of the American Chemical Society, vol. 134, pp. 19026–19034, 2012, doi: 10.1021/ja306320s
  7. H. Wang, E. Nakata, and I. Hamachi, “Recent Progress in Strategies for the Creation of Protein-Based Fluorescent Biosensors,” ChemBioChem, vol. 10, no. 16, pp. 2560–2577, Nov. 2009, doi: 10.1002/cbic.200900249
  8. V. Trümper et al., “Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells,” Theranostics, vol. 9, no. 19, pp. 5444–5463, 2019, doi: 10.7150/thno.29367
  9. D. Zhang, E. Redington, and Y. Gong, “Rational engineering of ratiometric calcium sensors with bright green and red fluorescent proteins,” Communications Biology, vol. 4, no. 1, Dec. 2021, doi: 10.1038/s42003-021-02452-z
  10. D. W. Piston and G. J. Kremers, “Fluorescent protein FRET: the good, the bad and the ugly,” Trends in Biochemical Sciences, vol. 32, no. 9, pp. 407–414, 2007, doi: 10.1016/j.tibs.2007.08.003
  11. D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov, “Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues,” Physiological Reviews, vol. 90, no. 3, pp. 1103–1163, Jul. 2010, doi: 10.1152/physrev.00038.2009
  12. N. C. Shaner et al., “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum,” Nat Methods, vol. 10, no. 5, pp. 407–409, 2013, doi: 10.1038/nmeth.2413.A
  13. I. Stockmar et al., “Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy,” Scientific Reports, vol. 8, no. 1, pp. 1–11, 201