Directed Evolution of an Angiopoietin-2 Ligand Trap by Somatic Hypermutation and Cell Surface Display*
| العنوان: | Directed Evolution of an Angiopoietin-2 Ligand Trap by Somatic Hypermutation and Cell Surface Display* |
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| المؤلفون: | Shikha Sharma, Julian E. Sale, Teonchit Nuamchit, Hiroshi Arakawa, Jean-Marie Buerstedde, Kathryn H. Steele, Nicholas P.J. Brindle |
| المصدر: | The Journal of Biological Chemistry |
| بيانات النشر: | American Society for Biochemistry and Molecular Biology, 2013. |
| سنة النشر: | 2013 |
| مصطلحات موضوعية: | Directed Evolution, Somatic hypermutation, Ligand Binding Protein, Plasma protein binding, Protein Engineering, Biochemistry, Receptor tyrosine kinase, Angiopoietin-2, 03 medical and health sciences, 0302 clinical medicine, Cell surface receptor, Humans, Molecular Biology, 030304 developmental biology, 0303 health sciences, Endothelial Cell, biology, HEK 293 cells, Cell Surface Receptor, Cell Biology, Angiopoietin receptor, Molecular biology, Receptor, TIE-2, Ligand-binding Protein, Cell biology, Protein Structure, Tertiary, HEK293 Cells, Ectodomain, 030220 oncology & carcinogenesis, Protein Structure and Folding, biology.protein, Angiogenesis, Directed Molecular Evolution, Protein Binding |
| الوصف: | Background: The ligand angiopoietin2 contributes to vascular diseases. Results: A new directed evolution method was used to create a specific angiopoietin2-binding protein from a nonspecific angiopoietin receptor. Conclusion: The receptor binding specificity can be switched with just three residue changes. Significance: The new protein has therapeutic potential, and the directed evolution method has advantages for evolving mammalian proteins. Tie2 is a receptor tyrosine kinase that is essential for the development and maintenance of blood vessels through binding the soluble ligands angiopoietin 1 (Ang1) and 2 (Ang2). Ang1 is constitutively produced by perivascular cells and is protective of the adult vasculature. Ang2 plays an important role in blood vessel formation and is normally expressed during development. However, its re-expression in disease states, including cancer and sepsis, results in destabilization of blood vessels contributing to the pathology of these conditions. Ang2 is thus an attractive therapeutic target. Here we report the directed evolution of a ligand trap for Ang2 by harnessing the B cell somatic hypermutation machinery and coupling this to selectable cell surface display of a Tie2 ectodomain. Directed evolution produced an unexpected combination of mutations resulting in loss of Ang1 binding but maintenance of Ang2 binding. A soluble form of the evolved ectodomain binds Ang2 but not Ang1. Furthermore, the soluble evolved ectodomain blocks Ang2 effects on endothelial cells without interfering with Ang1 activity. Our study has created a novel Ang2 ligand trap and provided proof of concept for combining surface display and exogenous gene diversification in B cells for evolution of a non-immunoglobulin target. |
| اللغة: | English |
| تدمد: | 1083-351X 0021-9258 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d1831936e20d8708d4a4802c0db100abTest http://europepmc.org/articles/PMC3829167Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....d1831936e20d8708d4a4802c0db100ab |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 1083351X 00219258 |
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