Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3
Abstract Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RT...
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Nature Portfolio
2025-01-01
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| Online Access: | https://doi.org/10.1038/s41598-025-86194-6 |
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| author | Alexander J. D. Snow Tharushi Wijesiriwardena Benjamin J. Lane Brendan Farrell Polly C. Dowdle Matilda Katan Stephen P. Muench Alexander L. Breeze |
| author_facet | Alexander J. D. Snow Tharushi Wijesiriwardena Benjamin J. Lane Brendan Farrell Polly C. Dowdle Matilda Katan Stephen P. Muench Alexander L. Breeze |
| author_sort | Alexander J. D. Snow |
| collection | DOAJ |
| description | Abstract Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 h. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 µg/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation (t < 0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-34e7374febc84d1789dca124d95e8a712025-01-26T12:32:46ZengNature PortfolioScientific Reports2045-23222025-01-011511910.1038/s41598-025-86194-6Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3Alexander J. D. Snow0Tharushi Wijesiriwardena1Benjamin J. Lane2Brendan Farrell3Polly C. Dowdle4Matilda Katan5Stephen P. Muench6Alexander L. Breeze7Astbury Centre for Structural Molecular Biology, University of LeedsAstbury Centre for Structural Molecular Biology, University of LeedsAstbury Centre for Structural Molecular Biology, University of LeedsAstbury Centre for Structural Molecular Biology, University of LeedsAstbury Centre for Structural Molecular Biology, University of LeedsInstitute of Structural and Molecular Biology, Division of Biosciences, University College LondonAstbury Centre for Structural Molecular Biology, University of LeedsAstbury Centre for Structural Molecular Biology, University of LeedsAbstract Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 h. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 µg/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation (t < 0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.https://doi.org/10.1038/s41598-025-86194-6Cell-freeFGFRRTKOncoproteinKinaseSMA |
| spellingShingle | Alexander J. D. Snow Tharushi Wijesiriwardena Benjamin J. Lane Brendan Farrell Polly C. Dowdle Matilda Katan Stephen P. Muench Alexander L. Breeze Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 Scientific Reports Cell-free FGFR RTK Oncoprotein Kinase SMA |
| title | Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 |
| title_full | Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 |
| title_fullStr | Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 |
| title_full_unstemmed | Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 |
| title_short | Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3 |
| title_sort | cell free expression and sma copolymer encapsulation of a functional receptor tyrosine kinase disease variant fgfr3 tacc3 |
| topic | Cell-free FGFR RTK Oncoprotein Kinase SMA |
| url | https://doi.org/10.1038/s41598-025-86194-6 |
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