Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess
With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O<sub>2</sub>, etc.). However...
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MDPI AG
2021-07-01
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| Series: | Sensors |
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| Online Access: | https://www.mdpi.com/1424-8220/21/15/4993 |
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| author | Xuerao Ning Takanobu Yasuda Tetsuya Kitaguchi Hiroshi Ueda |
| author_facet | Xuerao Ning Takanobu Yasuda Tetsuya Kitaguchi Hiroshi Ueda |
| author_sort | Xuerao Ning |
| collection | DOAJ |
| description | With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O<sub>2</sub>, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the <i>Brevibacillus</i> culture media. |
| first_indexed | 2024-03-10T09:09:18Z |
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| id | doaj.art-8f3ae14a6357453f874471a60773da0e |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-03-10T09:09:18Z |
| publishDate | 2021-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-8f3ae14a6357453f874471a60773da0e2023-11-22T06:08:52ZengMDPI AGSensors1424-82202021-07-012115499310.3390/s21154993Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in BioprocessXuerao Ning0Takanobu Yasuda1Tetsuya Kitaguchi2Hiroshi Ueda3Tokyo Institute of Technology, Graduate School of Life Science and Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Kanagawa, JapanTokyo Institute of Technology, Graduate School of Life Science and Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Kanagawa, JapanLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Kanagawa, JapanLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Kanagawa, JapanWith the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O<sub>2</sub>, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the <i>Brevibacillus</i> culture media.https://www.mdpi.com/1424-8220/21/15/4993His-tagfluorescent biosensorimmunoassayrecombinant protein production |
| spellingShingle | Xuerao Ning Takanobu Yasuda Tetsuya Kitaguchi Hiroshi Ueda Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess Sensors His-tag fluorescent biosensor immunoassay recombinant protein production |
| title | Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess |
| title_full | Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess |
| title_fullStr | Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess |
| title_full_unstemmed | Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess |
| title_short | Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess |
| title_sort | construction of fluorescent immunosensor quenchbody to detect his tagged recombinant proteins produced in bioprocess |
| topic | His-tag fluorescent biosensor immunoassay recombinant protein production |
| url | https://www.mdpi.com/1424-8220/21/15/4993 |
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