Abstract
This chapter describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination. Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His6-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.
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Acknowledgments
This work was supported by NIGMS Protein Structure Initiative grant 1U54 GM074901 (which supports CESG) and NIGMS grant P41 RR02301 (which supports the National Magnetic Resonance Facility at Madison, where NMR spectroscopy was carried out). Dr. Dmitriy A. Vinarov led initial work at CESG on the development of the wheat germ cell-free system for structural genomics efforts with assistance from Ms. Ejan Tyler and Dr. Carrie Loushin Newman. The authors thank Prof. Yaeta Endo and others at Ehime University for advice and encouragement and also the staff members of CellFree Sciences Ltd. (Yokohama, Japan) who made this approach commercially viable.
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Makino, Si., Goren, M.A., Fox, B.G., Markley, J.L. (2010). Cell-Free Protein Synthesis Technology in NMR High-Throughput Structure Determination. In: Endo, Y., Takai, K., Ueda, T. (eds) Cell-Free Protein Production. Methods in Molecular Biology, vol 607. Humana Press. https://doi.org/10.1007/978-1-60327-331-2_12
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DOI: https://doi.org/10.1007/978-1-60327-331-2_12
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