A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes
Abstract
:1. Introduction
2. Results and Discussion
2.1. Background
2.2. FR-Emitting Mutant Selection
2.3. BL Properties of RE-R215K with Firefly Luciferin
2.4. Bioluminescence Properties of R215K with 6′-Amino-Analogs and Akalumine
2.4.1. Bioluminescence Activity
2.4.2. Substrate KM and Luminescence Kinetics
2.4.3. Catalytic Constant and Efficiencies
2.4.4. Bioluminescence Spectra
2.4.5. Thermostability
2.5. Bioluminescence of Bacteria Expressing R215K with 6′-(1-pyrrolidinyl)luciferin
2.6. Structure and Function Relationships
2.7. Comparison RE-R215K/N5 with Other Beetle FR Emitting Luciferin-Luciferases
3. Material and Methods
3.1. Plasmids and Beetle Luciferases cDNAs
3.2. Site-Directed Mutagenesis
3.3. Luciferase Expression and Purification
3.4. Measurement of Luciferase Activity
3.5. Kinetics Measurements and KM Determination
3.6. Luciferyl-Adenylate Synthesis
3.7. Determination of kcat and kox
3.8. 6′-Substituted Amino Analogs
3.9. Bioluminescence Spectra
4. Concluding Remarks
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Luciferase | Specific Activity (109 cps/mg) * | Oxidative Activity (109 cps/mg) * | KM ATP (μM) | KM LH2 (μM) | KM N5 (μM) | kcat LH2 (cps) | kcat N5 (cps) | kcat/ KM LH2 (cps M−1) | kcat/ KM N5 (cps M−1) | kox/ KM ATP (cps M−1) | kox/ KM LH2 (cps M−1) |
---|---|---|---|---|---|---|---|---|---|---|---|
RE WT | 70 | 65 | 230 | 7 | 0.5 | 8.3 | 7.1 | 1.2 | 14.2 | 0.03 | 1.11 |
R215K | 89 | 83 | 50 | 40 | 1.0 | 10 | 9.4 | 0.25 | 9.4 | 0.19 | 0.23 |
Analogs | Wavelength (Half-Bandwidth) (nm) | Specific Activity (109 cps/mg) ** | kcat (10−6 cps) ** | |||
---|---|---|---|---|---|---|
RE WT * | R215K | RE WT | R215K | RE WT | R215K | |
LH2 | 626 (82) | 629 (74) | 86.3 | 104 | 9.6 | 11.7 |
NH2-LH | 612 (87) | 614 (83) | 33 | 31.6 | 3.7 | 3.5 |
Mor | 634 (92) | 644 (72) | 22.9 | 22.2 | 2.5 | 2.4 |
N5 | 644 (84) | 650 (81) | 63.4 | 81 | 7.1 | 9.0 |
N6 | 651 (84) | 656 (76) | 12.4 | 6.2 | 1.3 | 0.6 |
N7 | 637 (102) | 647 (82) | 34.2 | 8.6 | 3.8 | 0.9 |
AkaLumine | 681 (81) | 683 (85) | 0.9 | 0.4 | 0.1 | 0.05 |
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Viviani, V.R.; Bevilaqua, V.R.; de Souza, D.R.; Pelentir, G.F.; Kakiuchi, M.; Hirano, T. A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes. Int. J. Mol. Sci. 2021, 22, 303. https://doi.org/10.3390/ijms22010303
Viviani VR, Bevilaqua VR, de Souza DR, Pelentir GF, Kakiuchi M, Hirano T. A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes. International Journal of Molecular Sciences. 2021; 22(1):303. https://doi.org/10.3390/ijms22010303
Chicago/Turabian StyleViviani, Vadim R., Vanessa R. Bevilaqua, Daniel R. de Souza, Gabriel F. Pelentir, Michio Kakiuchi, and Takashi Hirano. 2021. "A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes" International Journal of Molecular Sciences 22, no. 1: 303. https://doi.org/10.3390/ijms22010303