Expression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cells
| Autor: | Sasajima, Yoshiyuki, Iwasaki, Ryohei, Tsumoto, Kouhei, Kumagai, Izumi, Ihara, Masaki, Ueda, Hiroshi |
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| Rok vydání: | 2010 |
| Předmět: |
Recombinant Fusion Proteins
Immunology Blotting Western Molecular Sequence Data Immunoglobulin Variable Region Enzyme-Linked Immunosorbent Assay Transfection Antigen Chlorocebus aethiops medicine Immunology and Allergy Animals Humans Amino Acid Sequence Cell fusion biology medicine.diagnostic_test Alkaline Phosphatase Fusion protein Molecular biology Blot Kinetics Placental alkaline phosphatase Biochemistry Immunoassay COS Cells biology.protein Alkaline phosphatase Antibody Plasmids |
| Zdroj: | Journal of immunological methods. 361(1-2) |
| ISSN: | 1872-7905 |
| Popis: | Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional VH-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent VH/VL interaction for antigen quantitation. |
| Databáze: | OpenAIRE |
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