Expression of single-chain antibody-barstar fusion in plants

• Published in: Biochimie Vol. 89; no. 1; pp. 31 - 38
• Main Authors: Semenyuk, Ekaterina G., Stremovskiy, Oleg A., Edelweiss, Evelina F., Shirshikova, Olga V., Balandin, Taras G., Buryanov, Yaroslav I., Deyev, Sergey M.
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 2007
• Subjects: Bacterial Proteins - genetics; Barnase; Barstar; Blotting, Western; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; ErbB Receptors - immunology; Flow Cytometry; HER1 (EGFR); Humans; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - immunology; Microscopy, Fluorescence; Nicotiana - genetics; Plants, Genetically Modified; Protein Engineering - methods; Receptor, ErbB-2; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - immunology; Single-chain Fv antibody; Transgenic plants; TSP; Barnase; IMAC; Single-chain Fv antibody; scFv; HRP; Barstar; Transgenic plants; HER1 (EGFR); ELISA
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2006.07.012

We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar–barnase system. Based on barstar–barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.