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RESEARCH PRODUCT
Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells
Sanna T.h. UotilaMinna KulmalaMarkku S. KulomaaOlli H. LaitinenHenri R. NordlundNisse Kalkkinensubject
StreptavidinBiotin bindingRecombinant Fusion ProteinsGreen Fluorescent ProteinsBiophysicsBiotinEnzyme-Linked Immunosorbent AssayNanotechnologySpodopteraBiologyBiochemistryChromatography AffinityGreen fluorescent protein03 medical and health scienceschemistry.chemical_compoundBiotinAnimalsMolecular BiologyDNA PrimersPlant Proteins030304 developmental biology0303 health sciencesInsect cellDownstream processingBase Sequence030302 biochemistry & molecular biologyCell BiologyAllergensAntigens PlantFusion proteinFluorescencechemistryBiochemistryBaculoviridaedescription
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.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2005-10-01 | Biochemical and Biophysical Research Communications |