1. Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells
- Author
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Makoto Takeuchi, Atsushi Tamai, Shinya Ohki, Masashi Mori, and Koji Dohi
- Subjects
Calmodulin ,Genetic Vectors ,medicine.disease_cause ,Transfection ,Biochemistry ,law.invention ,Aprotinin ,law ,Dihydrofolate reductase ,Tobacco ,medicine ,Phosphoprotein Phosphatases ,Disulfides ,Protein kinase A ,Escherichia coli ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Cells, Cultured ,stable-isotope labeling ,biology ,Molecular mass ,Chemistry ,BY―2 ,Inducible virus vector ,Molecular biology ,Recombinant Proteins ,Tetrahydrofolate Dehydrogenase ,Agrobacterium tumefaciens ,Isotope Labeling ,biology.protein ,Recombinant DNA ,Heteronuclear single quantum coherence spectroscopy - Abstract
We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg ^N-labeled protein suitable for NMR experiments. The ^1H-^N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.
- Published
- 2008