title: Expression and characterization of a functional single-chain variable fragment (scFv) protein recognizing MCF7 breast cancer cells in E. coli cytoplasm.
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.
authors.affiliation: Department of Microbiology and Immunology, Faculty of Medicine and health sciences, Alneelain University, P.O. Box 12702, Khartoum, Sudan. ilhamomer@yahoo.com
authors.forename: Ilham Omer
authors.initials: IO
authors.lastname: Mahgoub
mesh_terms: D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000235:genetics*; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors / Q000276:immunology; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D000939:Epitopes; D057127:Single-Chain Antibodies; D066246:ErbB Receptors