Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells
| Autor: | Sanna T.H. Uotila, Minna Kulmala, Markku S. Kulomaa, Olli H. Laitinen, Henri R. Nordlund, Nisse Kalkkinen |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Streptavidin
Biotin binding Recombinant Fusion Proteins Green Fluorescent Proteins Biophysics Biotin Enzyme-Linked Immunosorbent Assay Nanotechnology Spodoptera Biology Biochemistry Chromatography Affinity Green fluorescent protein 03 medical and health sciences chemistry.chemical_compound Animals Molecular Biology DNA Primers Plant Proteins 030304 developmental biology 0303 health sciences Insect cell Downstream processing Base Sequence 030302 biochemistry & molecular biology Cell Biology Allergens Antigens Plant Fusion protein Fluorescence chemistry Baculoviridae |
| Zdroj: | Biochemical and Biophysical Research Communications. 336:232-238 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2005.08.095 |
| Popis: | The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches. |
| Databáze: | OpenAIRE |
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