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Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

Abstract

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein–protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16–40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5–6 d for plasmid construction and 3–4 d for incorporating the non-natural amino acids into proteins.

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Figure 1: Plasmid maps.
Figure 2
Figure 3: Incorporation of L-tyrosine and IY into the human Src kinase.
Figure 4: Analysis of the efficiency of the amber suppression incorporating pBpa.
Figure 5: In vivo photo-cross-linking between the Grb2 protein with pBpa at position 171 and endogenous proteins in CHO T-REx cells.

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Acknowledgements

This work was suported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, and the Ministry of Education, Culture, Science and Technology of Japan.

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Correspondence to Shigeyuki Yokoyama.

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Hino, N., Hayashi, A., Sakamoto, K. et al. Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code. Nat Protoc 1, 2957–2962 (2006). https://doi.org/10.1038/nprot.2006.424

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