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Cell-free protein production and labeling protocol for NMR-based structural proteomics

Nature Methods volume 1, pages 149–153 (2004)Cite this article

Abstract

Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 μl) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate 15N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with 13C,15N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the β-grasp protein family.

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Figure 1
Figure 2: Cell-free expression and purification of [U-13C,15N]Ntag-At3g01050.1.
Figure 3: NMR structure of At3g01050.1.

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Protein Data Bank

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Acknowledgements

We thank Y. Endo and CellFree Sciences Co., Ltd., for advice and encouragement in using the wheat germ cell-free system that they have developed; and M. Kainosho for sharing the unpublished information cited here. This research was supported by a grant from the National Institutes of Health Protein Structure Initiative (1 P50 GM64598 to J.L.M.).

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Authors and Affiliations

  1. Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, 433 Babcock Drive, Madison, 53706, Wisconsin, USA

    Dmitriy A Vinarov, Betsy L Lytle, Francis C Peterson, Ejan M Tyler, Brian F Volkman & John L Markley

  2. Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, 53706, Wisconsin, USA

    Dmitriy A Vinarov, Ejan M Tyler & John L Markley

  3. Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, Wisconsin, USA

    Betsy L Lytle, Francis C Peterson & Brian F Volkman

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  1. Dmitriy A Vinarov
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Correspondence to John L Markley.

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Competing interests

As stated in the article, the Center for Eukaryotic Structural Genomics and the University of Wisconsin–Madison have a collaborative agreement with Ehime University and Cell-Free Sciences, Co. Ltd., a company located in Yokohama, Japan. Under the terms of this agreement, the parties involved are codeveloping and improving the wheat germ cell-free technology. Discussions are ongoing concerning the establishment of a US company to further develop and commercialize this technology; this company might involve certain authors of this paper (D.A.V. and J.L.M.).

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Vinarov, D., Lytle, B., Peterson, F. et al. Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1, 149–153 (2004). https://doi.org/10.1038/nmeth716

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