Cell-free protein synthesis offers fast and inexpensive access to selectively isotope labeled proteins thath can be measured by NMR spectroscopy in the presence of all the unlabeled proteins in the reaction mixture. No chromatographic purification is required. Using an extract from Escherichia coli in a simple dialysis system, the target protein can be prepared at a typical concentration of about 1 mg/ml, which is sufficient for subsequent analysis by NMR. This chapter describes in detail the protocol used in the authors' laboratory.
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Acknowledgments
The authors thank Nikki Moreland and Karin Loscha for a critical reading of the manuscript and Peter S. C. Wu for useful discussions. This work was supported by the Australian Research Council, including project grants (to G. O. and N. E. D.), a CSIRO-Linkage Fellowship (to K. O.) and a Federation Fellowship (to G. O.).
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Apponyi, M.A., Ozawa, K., Dixon, N.E., Otting, G. (2008). Cell-Free Protein Synthesis for Analysis by NMR Spectroscopy. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_16
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