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
Title:
Transmembrane signaling on a protocell: creation of receptor-enzyme chimeras for immunodetection of specific antibodies and antigens
Other Titles:
Scientific Reports
Publication Date:
03 December 2019
Citation:
Su, J., Kitaguchi, T., Ohmuro-Matsuyama, Y. et al. Transmembrane signaling on a protocell: Creation of receptor-enzyme chimeras for immunodetection of specific antibodies and antigens. Sci Rep 9, 18189 (2019). https://doi.org/10.1038/s41598-019-54539-7
Abstract:
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-IV5m protein expressed in protocells, suggesting utility of this platform for detection of diverse antigen types.
License type:
http://creativecommons.org/licenses/by/4.0/
Funding Info:
This work was supported partly by Strategic International Collaborative Research Program (SICORP) and JST-Mirai Program (Grant Number JPMJMI18D9) under Japan Science and Technology Agency, Japan, by JSPS KAKENHI (Grant Numbers JP15H04191, JP18H03851) under the Japan Society for the Promotion of Science, Japan, and Special Research Grant from the Nakatani Foundation, Japan. J.S. was supported by ACEEES Program, Tokyo Institute of Technology.
Description:
ISSN:
2045-2322
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