Abstract
Escherichia coli cell extract-based coupled transcription–translation cell-free system has been developed for large-scale production of protein samples for both X-ray crystallography (selenomethionine substitution) and NMR (stable-isotope labeling). For both cases, higher labeling/substitution efficiency can be achieved compared with the production using cell-based expression system. In addition, as the system is easily adapted to automated and/or high-throughput procedures, it is an especially suitable protein expression method for structural genomics and proteomics project. In this chapter, the procedure for large-scale protein production for structure determination using our E. coli cell-free system is presented.
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Acknowledgments
The author would like to thank Drs. Takashi Yabuki, Takayoshi Matsuda, and Makoto Inoue for development of our cell-free system.
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Kigawa, T. (2010). Cell-Free Protein Production System with the E. coli Crude Extract for Determination of Protein Folds. 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_10
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DOI: https://doi.org/10.1007/978-1-60327-331-2_10
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