Selection, purification, and characterization of a HER2-targeting soluble designed ankyrin repeat protein by E. coli surface display using HER2-positive melanoma cells
| Autor: | Wenbing Yao, Yuting Yi, Xiaofei Chen, Xiangdong Gao, Li Liu, Xiaoxiao Yu, Xiaoda Song |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 0301 basic medicine Receptor ErbB-2 medicine.drug_class Antineoplastic Agents Monoclonal antibody Biochemistry Viral Proteins 03 medical and health sciences 0302 clinical medicine Trastuzumab Cell Line Tumor Escherichia coli medicine Humans MTT assay Amino Acid Sequence skin and connective tissue diseases Melanoma Cell Proliferation Gene Library Antibody mimetic biology Chemistry General Medicine biology.organism_classification medicine.disease Molecular biology Ankyrin Repeat 030104 developmental biology DARPin biology.protein Antibody Mimiviridae Bacteria Protein Binding 030215 immunology Biotechnology medicine.drug |
| Zdroj: | Preparative Biochemistry & Biotechnology. 48:144-150 |
| ISSN: | 1532-2297 1082-6068 |
| Popis: | Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells. DARPin selected from the library could be expressed in soluble form with a yield of 70 mg/L. After purified by two continuous and easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC. Selected DARPin showed significant HER2-targeting ability with an affinity of 1.05 ± 0.47 µM. MTT assay demonstrated that at the concentration of 640 nM, the selected DARPin dimer could inhibit the SK-BR-3 growth at a rate of 36.63 and 46.34% in 48 and 72 hr incubation separately, which was similar to trastuzumab (43.12 and 49.14% separately). These findings suggested that it was an effective method to select antibody mimetic DARPin by bacteria surface display combined with live cells sorting and provided a drug candidate for cancer therapy. |
| Databáze: | OpenAIRE |
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