Structure-based design of covalent nanobody binders for a thermostable green fluorescence protein
The use of green fluorescence protein (GFP) has advanced numerous areas of life sciences. An ultra-thermostable GFP (TGP), engineered from a coral GFP, offers potential advantages over traditional jellyfish-derived GFP because of its high stability. However, owing to its later discovery, TGP lacks t...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
China Science Publishing & Media Ltd.
2024-12-01
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| Series: | Acta Biochimica et Biophysica Sinica |
| Subjects: | |
| Online Access: | https://www.sciengine.com/doi/10.3724/abbs.2024233 |
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| Summary: | The use of green fluorescence protein (GFP) has advanced numerous areas of life sciences. An ultra-thermostable GFP (TGP), engineered from a coral GFP, offers potential advantages over traditional jellyfish-derived GFP because of its high stability. However, owing to its later discovery, TGP lacks the extensive toolsets available for GFP, such as heavy chain-only antibody binders known as nanobodies. In this study, we report the crystal structure of TGP in complex with Sb92, a synthetic nanobody identified from a previous in vitro screening, revealing Sb92’s precise three-dimensional epitope. This structural insight, alongside the previously characterized Sb44-TGP complex, allows us to rationally design disulfide bonds between the antigen and the antibody for tighter interactions. Using biochemical analysis, we identify two bridged complexes (TGP A18C-Sb44 V100C and TGP E118C-Sb92 S57C), with the TGP-Sb92 disulfide pair showing high resistance to reducing agents. Our study expands the toolkit available for TGP and should encourage its wider applications. |
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| ISSN: | 1672-9145 |