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Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells

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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 15N-labeled protein suitable for NMR experiments. The 1H-15N 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.

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Abbreviations

BPTI:

Bovine pancreatic trypsin inhibitor

BY-2:

Nicotiana tabacum cv. Bright Yellow 2

CPI-17(22-120):

Functional domain of 17-kDa protein phosphatase-1 inhibitor protein

CaM:

Calmodulin

DHFR:

Dihydrofolate reductase

ER:

Endoplasmic reticulum

HSQC:

Heteronuclear single quantum correlation

NMR:

Nuclear magnetic resonance

ToMV:

Tomato mosaic virus

XVE:

Chimeric LexA-VP16-ER transactivator

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Acknowledgements

The plasmid for estradiol-inducible gene expression was kindly provided by Dr. Nam-Hai Chua of Rockefeller University. The authors thank technicians at the Center for Nano Materials and Technology, JAIST for maintenance of the 750 MHz NMR machine used in this study. Part of this project was supported by Kyoto-Advanced Nanotechnology Network operated in “Nanotechnology Network”, promoted by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. The work was financially supported in part by a Grant-in-Aid (#20510198 to S.O. and M.M.) from MEXT, Japan. M. M. is a grant holder of JST-CREST, Japan.

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Authors and Affiliations

  1. Japan Advanced Institute of Science and Technology (JAIST), Center for Nano Materials and Technology (CNMT), 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan

    Shinya Ohki & Makoto Takeuchi

  2. Laboratory of Plant Gene Technology, Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan

    Koji Dohi, Atsushi Tamai & Masashi Mori

  3. CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, 322-0012, Japan

    Koji Dohi & Masashi Mori

Authors
  1. Shinya Ohki
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  2. Koji Dohi
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  3. Atsushi Tamai
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  4. Makoto Takeuchi
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  5. Masashi Mori
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Correspondence to Shinya Ohki.

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Ohki, S., Dohi, K., Tamai, A. et al. Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells. J Biomol NMR 42, 271–277 (2008). https://doi.org/10.1007/s10858-008-9283-x

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  • DOI: https://doi.org/10.1007/s10858-008-9283-x

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