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

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Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-03-08 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00149
Setsuko Tsuboi ₁ , Takashi Jin _{1,

2}

Affiliation

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.

中文翻译：

重组蛋白（荧光素酶-IgG结合域）共轭量子点，用于表皮生长因子受体的BRET耦合近红外成像

为了对癌细胞上表达的表皮生长因子受体（EGFR）进行高灵敏的近红外（NIR）光学检测，通过直接标记带标签的海肾荧光素酶来制备生物发光共振能量转移（BRET）耦合的NIR量子点（QD）。（RLuc）重组蛋白（HisRLuc·GB1）修饰为谷胱甘肽包覆的CdSeTe / CdS QD（GSH-QDs）。重组蛋白具有两个功能基团，分别由荧光素酶和蛋白G的免疫球蛋白结合域（GB1）组成。重组蛋白（HisRLuc·GB1）结合的QD（GB1·RLuc-QDs）显示出BRET偶联的近红外发射，这是由于从萤光素到QDs的能量转移，BRET效率高达约 50％。由于GB1·RLuc-QD在其表面具有GB1结构域，所以QD具有结合免疫球蛋白G（IgG）的Fc部分的能力。所得的结合IgG的QD可用作具有NIR荧光和BRET耦合NIR发射的分子成像探针。为了对癌细胞上的EGFR进行NIR光学检测，我们将抗EGFR单克隆抗体与GB1·RLuc-QDs偶联。在此处，我们表明，通过GB1·RLuc-QD的BRET耦合NIR发射对EGFR的检测灵敏度至少比QD的NIR荧光检测灵敏度高三倍。抗EGFR抗体与GB1·RLuc-QD之间的结合物使得在活细胞中对EGFR进行基于BRET的高灵敏度NIR成像成为可能。

更新日期：2018-03-08

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