Transmembrane signaling on a protocell: Creation of receptor-enzyme chimeras for immunodetection of specific antibodies and antigens
Transmembrane signaling on a protocell: Creation of receptor-enzyme chimeras for immunodetection of specific antibodies and antigens
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DOI：
10.1038/s41598-019-54539-7
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发表时间：
2019-12
影响因子：
4.6
通讯作者：
Jiulong Su;T. Kitaguchi;Y. Ohmuro-Matsuyama;Theresa Seah;F. Ghadessy;Shawn Hoon;H. Ueda
中科院分区：
文献类型：
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作者：
Jiulong Su;T. Kitaguchi;Y. Ohmuro-Matsuyama;Theresa Seah;F. Ghadessy;Shawn Hoon;H. Ueda
众所周知，对许多小室中产生的荧光信号进行数字计数可以显着提高酶联免疫吸附测定（ELISA）的检测灵敏度。然而，报道的数字 ELISA 系统需要大量的洗涤步骤来消除背景信号，这会影响其性能。为了解决这个问题，我们开发了一种囊泡（Protocell）阵列，其中外部蛋白质分析物的结合与信号放大和囊泡内荧光读出相结合。我们选择β-葡萄糖醛酸酶（GUS）作为报告酶，因为其功能需要通过一对二聚体的二聚化来组装四个亚基，而这对二聚体可以被一组界面突变抑制。使用热稳定的GUS突变体IV-5，我们筛选出界面突变体（M516K，F517W）来创建IV5m-一种具有高热稳定性和诱导二聚化条件活性的突变体。在连接短的 N 末端标签和跨膜 (TM) 序列后，融合蛋白通过原始细胞内的无细胞蛋白合成来表达。当在原始细胞外部应用相应的标签特异性抗体时，通过添加荧光底物，在囊泡内部观察到 GUS 活性明显增加，这可能是由于标记的 TM 蛋白自发整合到囊泡中，并通过与展示的标签结合的抗体进行二聚化。此外，使用流式细胞术，通过对暴露于不同浓度的外部抗体（包括曲妥珠单抗）的荧光原始细胞进行计数来获得定量数字读数。此外，通过使用抗咖啡因 VHH-SpyCatcher 融合蛋白，可以使用在原始细胞中表达的 SpyTag 融合 TM-IV5m 蛋白来检测咖啡因，这表明该平台可用于检测不同抗原类型。
It is known that digital counting of fluorescent signals generated in many small compartments can significantly improve the detection sensitivity of the enzyme-linked immunosorbent assay (ELISA). However, the reported digital ELISA systems need extensive washing steps to remove background signal, which hampers their performance. To tackle this problem, we developed a vesicle (Protocell) array wherein binding of an external protein analyte is coupled to signal amplification and intra-vesicular fluorescence readout. We chose β-glucuronidase (GUS) as a reporter enzyme as its function requires assembly of four subunits through dimerization of a pair of dimers that can be inhibited by a set of interface mutations. Using a thermostabilized GUS mutant IV-5, we screened out an interface mutant (M516K, F517W) to create IV5m- a mutant with high thermostability and activity conditional on induced dimerization. After tethering a short N-terminal tag and transmembrane (TM) sequences, the fusion protein was expressed by cell-free protein synthesis inside protocells. When a corresponding tag-specific antibody was applied outside of the protocells, a clear increase in GUS activity was observed inside vesicles by adding fluorescent substrate, probably due to spontaneous integration of the tagged TM protein into the vesicles and dimerization by the antibody bound to the displayed tag. Furthermore, using flow cytometry, quantitative digital read out was obtained by counting fluorescent protocells exposed to varying concentrations of external antibodies that included Trastuzumab. Additionally, through use of an anti-caffeine VHH-SpyCatcher fusion protein, caffeine could be detected using SpyTag-fused TM-IV5mprotein expressed in protocells, suggesting utility of this platform for detection of diverse antigen types.