Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis
<i>Gaussia</i> luciferase (<i>G</i>Luc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly e...
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MDPI AG
2024-11-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/14/11/528 |
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| author | Vinayakumar Gedi Eun Hye Kim Bohyun Oh Young-Pil Kim |
| author_facet | Vinayakumar Gedi Eun Hye Kim Bohyun Oh Young-Pil Kim |
| author_sort | Vinayakumar Gedi |
| collection | DOAJ |
| description | <i>Gaussia</i> luciferase (<i>G</i>Luc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type <i>G</i>Luc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in <i>G</i>Luc (designated as <i>G</i>Luc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the <i>G</i>Luc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in <i>G</i>Luc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications. |
| format | Article |
| id | doaj-art-d11470965add44eebda8203072de84bb |
| institution | OA Journals |
| issn | 2079-6374 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj-art-d11470965add44eebda8203072de84bb2025-08-20T02:08:08ZengMDPI AGBiosensors2079-63742024-11-01141152810.3390/bios14110528Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided MutagenesisVinayakumar Gedi0Eun Hye Kim1Bohyun Oh2Young-Pil Kim3Department of Life Science, Hanyang University, Seoul 04763, Republic of KoreaDepartment of Life Science, Hanyang University, Seoul 04763, Republic of KoreaDepartment of Life Science, Hanyang University, Seoul 04763, Republic of KoreaDepartment of Life Science, Hanyang University, Seoul 04763, Republic of Korea<i>Gaussia</i> luciferase (<i>G</i>Luc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type <i>G</i>Luc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in <i>G</i>Luc (designated as <i>G</i>Luc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the <i>G</i>Luc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in <i>G</i>Luc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications.https://www.mdpi.com/2079-6374/14/11/528bioluminescence<i>Gaussia</i> luciferasemutagenesiscoelenterazinereporter |
| spellingShingle | Vinayakumar Gedi Eun Hye Kim Bohyun Oh Young-Pil Kim Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis Biosensors bioluminescence <i>Gaussia</i> luciferase mutagenesis coelenterazine reporter |
| title | Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis |
| title_full | Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis |
| title_fullStr | Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis |
| title_full_unstemmed | Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis |
| title_short | Advanced Bioluminescence Reporter with Engineered <i>Gaussia</i> Luciferase via Sequence-Guided Mutagenesis |
| title_sort | advanced bioluminescence reporter with engineered i gaussia i luciferase via sequence guided mutagenesis |
| topic | bioluminescence <i>Gaussia</i> luciferase mutagenesis coelenterazine reporter |
| url | https://www.mdpi.com/2079-6374/14/11/528 |
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