Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein
Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In...
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| Format: | Article |
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MDPI AG
2024-01-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/14/1/54 |
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| author | Hao Zhang Xiaodong Tian Jing Zhang Hui-wang Ai |
| author_facet | Hao Zhang Xiaodong Tian Jing Zhang Hui-wang Ai |
| author_sort | Hao Zhang |
| collection | DOAJ |
| description | Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both <i>E. coli</i> and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness. |
| first_indexed | 2024-03-08T11:03:04Z |
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| id | doaj.art-2d11a80b9d83441fac9de60dd4689e53 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-08T11:03:04Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-2d11a80b9d83441fac9de60dd4689e532024-01-26T15:23:24ZengMDPI AGBiosensors2079-63742024-01-011415410.3390/bios14010054Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent ProteinHao Zhang0Xiaodong Tian1Jing Zhang2Hui-wang Ai3Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USACenter for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USACenter for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USACenter for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USAIntroducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both <i>E. coli</i> and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness.https://www.mdpi.com/2079-6374/14/1/54nonconical amino acidprotein engineeringredox sensitivityred-shifted fluorescence |
| spellingShingle | Hao Zhang Xiaodong Tian Jing Zhang Hui-wang Ai Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein Biosensors nonconical amino acid protein engineering redox sensitivity red-shifted fluorescence |
| title | Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein |
| title_full | Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein |
| title_fullStr | Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein |
| title_full_unstemmed | Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein |
| title_short | Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein |
| title_sort | engineering and characterization of 3 aminotyrosine derived red fluorescent variants of circularly permutated green fluorescent protein |
| topic | nonconical amino acid protein engineering redox sensitivity red-shifted fluorescence |
| url | https://www.mdpi.com/2079-6374/14/1/54 |
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