Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm |
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Authors: | Ilham Omer Mahgoub |
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Institution: | Department of Microbiology and Immunology, Faculty of Medicine and health sciences, Alneelain University, P.O. Box 12702, Khartoum, Sudan. ilhamomer@yahoo.com |
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Abstract: | Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production. |
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