Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs

Kumar, Manoj; Kovalski, Letícia; Broyles, David; Hunt, Eric A.; Daftarian, Pirouz; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K.

doi:10.1016/j.ab.2015.11.028

Cited by 4 publications

(2 citation statements)

References 39 publications

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“…In the GB1·RLuc-QDs + CTZ system, the emission intensity at 485 nm decreased two times compared to the emission intensity of HisRLuc·GB1 + CTZ. The efficiency of BRET from CTZ to QD can be calculated by the following equation: E = 1 – ( I DA /I D ) × 100%, where I D and I DA represent the emission intensity of the donor (CTZ) in the absence and presence of acceptor (QD), respectively. ,, In the GB1·RLuc-QDs + CTZ system, strong NIR emission (at ca. 830 nm) coupled by BRET was observed with a BRET efficiency of ca.…”

Section: Resultssupporting

confidence: 58%

“…Compared with the emission spectrum of HisRLuc•GB1 + CTZ, the spectrum of GB1•RLuc-QDs + CTZ shows a dual emission at 486 and 830 nm, which results from the BRET of CTZ to QDs (Figure 3c). In the GB1•RLuc-QDs + CTZ system, the emission intensity at 485 nm decreased two times 49,50 In the GB1•RLuc-QDs + CTZ system, strong NIR emission (at ca. 830 nm) coupled by BRET was observed with a BRET efficiency of ca.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors

Tsuboi¹

2018

Bioconjugate Chem.

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For the highly sensitive near-infrared (NIR) optical detection of epidermal growth factor receptors (EGFRs) expressed on cancer cells, bioluminescence resonance energy transfer (BRET) coupled NIR quantum dots (QDs) are prepared by direct conjugation of his-tagged Renilla luciferase (RLuc) recombinant protein (HisRLuc·GB1) to glutathione-coated CdSeTe/CdS QDs (GSH-QDs). The recombinant protein has two functional groups consisting of a luciferase enzyme and an immunoglobulin binding domain (GB1) of protein G. Recombinant protein (HisRLuc·GB1) conjugated QDs (GB1·RLuc-QDs) show BRET-coupled NIR emission, which results from energy transfer from luciferin to QDs with a high BRET efficiency of ca. 50%. Since the GB1·RLuc-QDs have the GB1 domain at their surface, the QDs have an ability to bind the Fc moiety of immunoglobulin G (IgG). The resulting IgG bound QDs can be used as a molecular imaging probe with NIR fluorescence and BRET-coupled NIR emission. For NIR optical detection of EGFRs on cancer cells, we conjugated anti-EGFR monoclonal antibody to the GB1·RLuc-QDs. Herein, we show that the detection sensitivity of EGFRs by BRET-coupled NIR emission of GB1·RLuc-QDs is at least three times higher than that of the NIR fluorescence of the QDs. The conjugates between anti-EGFR antibody and GB1·RLuc-QDs make it possible to perform BRET-based highly sensitive NIR imaging of EGFRs in living cells.

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Section: Resultssupporting

confidence: 58%

Section: ■ Results and Discussionmentioning

confidence: 99%

Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors

Tsuboi¹

2018

Bioconjugate Chem.

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Recent advances in homogenous immunoassays based on resonance energy transfer

Takkinen

Žvirblienė

2019

Current Opinion in Biotechnology

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Expression of a soluble truncated Vargula luciferase in Escherichia coli

Hunt

Moutsiopoulou

Broyles

et al. 2017

Protein Expression and Purification

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Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.

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Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs

Cited by 4 publications

References 39 publications

Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors

Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors

Recent advances in homogenous immunoassays based on resonance energy transfer

Expression of a soluble truncated Vargula luciferase in Escherichia coli

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