Abstract
Cell-free protein synthesis (CFPS) offers a fast and inexpensive means to incorporate unnatural amino acids (UAAs) site specifically into proteins. This enables engineering of proteins and allows production of protein-based probes for analysis of their interactions with other molecules. Using dialysis Escherichia coli CFPS system in combination with aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii high expression yield of proteins with site specifically incorporated UAAs can be achieved. Typically the target protein can be prepared at concentrations of about 1 mg/mL, which is generally sufficient for subsequent applications.
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Acknowledgements
This work has been supported by Profs. Gottfried Otting (Australian National University, Canberra, Australia) and Nicholas E. Dixon (University of Wollongong, Wollongong, Australia) and the Australian Research Council, including a fellowship to K.O. K.O. thanks Dr. Andrew V. Kralicek (Plant and Food Research, Auckland, New Zealand) for critical reading of an earlier version of the manuscript.
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Ozawa, K., Loh, C.T. (2014). Site-Specific Incorporation of Unnatural Amino Acids into Proteins by Cell-Free Protein Synthesis. In: Alexandrov, K., Johnston, W. (eds) Cell-Free Protein Synthesis. Methods in Molecular Biology, vol 1118. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-782-2_12
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DOI: https://doi.org/10.1007/978-1-62703-782-2_12
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