Structure‐based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity
| Autor: | Şeyma Işık, Aylin Ozdemir Bahadir, Beytullah Özgür, Melis Denizci Öncü, Gizem Dinler Doganay, Bertan Koray Balcioglu, Hasan Umit Ozturk, Muge Serhatli, Berrin Erdağ |
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| Rok vydání: | 2021 |
| Předmět: |
Vascular Endothelial Growth Factor A
medicine.drug_class Angiogenesis Biomedical Engineering chemical and pharmacologic phenomena Bioengineering Complementarity determining region Monoclonal antibody Applied Microbiology and Biotechnology In vivo Drug Discovery Escherichia coli Human Umbilical Vein Endothelial Cells medicine Humans Amino Acid Sequence Surface plasmon resonance biology Chemistry Process Chemistry and Technology Biological activity Kinase insert domain receptor General Medicine respiratory system Vascular Endothelial Growth Factor Receptor-2 Biochemistry biology.protein Molecular Medicine Antibody Single-Chain Antibodies Biotechnology |
| Zdroj: | Biotechnology and Applied Biochemistry. 69:2122-2137 |
| ISSN: | 1470-8744 0885-4513 |
| DOI: | 10.1002/bab.2273 |
| Popis: | Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR-2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single-chain variable fragment (scFv) against VEGFR-2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR-2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity. |
| Databáze: | OpenAIRE |
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