Abstract
Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-l-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein–protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed for pBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein–protein interactions of the target proteins at work.
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Acknowledgments
We thank Dr. Peter G. Schultz for providing the p-6xtRNA plasmids. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and a grant from the Japan Science and Technology Corporation (JST).
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Shiota, T., Nishikawa, Si., Endo, T. (2013). Analyses of Protein–Protein Interactions by In Vivo Photocrosslinking in Budding Yeast. In: Rapaport, D., Herrmann, J. (eds) Membrane Biogenesis. Methods in Molecular Biology, vol 1033. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-487-6_14
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DOI: https://doi.org/10.1007/978-1-62703-487-6_14
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-486-9
Online ISBN: 978-1-62703-487-6
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