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Incorporation of Unnatural Non-α-Amino Acids into the N-Terminus of Proteins in a Cell-Free Translation System

  • Takahiro HohsakaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

Unnatural amino acid mutagenesis allows us to introduce unnatural α-amino acids into internal positions of proteins in response to expanded codons such as amber and four-base codons. To improve the unnatural amino acid mutagenesis, the incorporation of unnatural α-amino acids and non-α-amino acids into the N-terminus of proteins has been achieved using expanded initiation codons. Here, we describe the method for the incorporation of fluorescent-labeled non-α-amino acids into the N-terminus of proteins in a cell-free translation system.

Key words

Unnatural amino acid Translation initiation Cell-free translation Chemical aminoacylation N-terminal labeling Fluorescence labeling 

Notes

Acknowledgments

This work was supported by Grants-in-Aid for Exploratory Research (17651122) and Scientific Research on Innovative Areas (20107005) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

References

  1. 1.
    Kudlicki W, Odom O W, Kramer G, Hardesty B (1994) Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese: Analysis by fluorescence. J Mol Biol 244, 319–331.PubMedCrossRefGoogle Scholar
  2. 2.
    Ramachandiran V, Willms C, Kramer G, Hardesty B (2000) Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome. J Biol Chem 275, 1781–1786.PubMedCrossRefGoogle Scholar
  3. 3.
    McIntosh B, Ramachandiran V, Kramer G, Hardesty B (2000) Initiation of protein synthesis with fluorophore-Met-tRNAf and the involvement of IF-2. Biochimie 82, 167–174.PubMedCrossRefGoogle Scholar
  4. 4.
    Gite S, Mamaev S, Olejnik J, Rothschild K (2000) Ultrasensitive fluorescence-based detection of nascent proteins in gels. Anal Biochem 279, 218–225.PubMedCrossRefGoogle Scholar
  5. 5.
    Taki M, Sawata S Y, Taira K (2001) Specific N-terminal biotinylation of a protein in vitro by a chemically modified tRNAfmet can support the native activity of the translated protein. J Biosci Bioeng 92, 149–153.PubMedCrossRefGoogle Scholar
  6. 6.
    Varshney U, RajBhandary U L (1990) Initiation of protein synthesis from a termination codon. Proc Natl Acad Sci USA 87, 1586–1590.PubMedCrossRefGoogle Scholar
  7. 7.
    Drabkin HJ, RajBhandary U L (1998) Initiation of protein synthesis in mammalian cells with codons other than AUG and amino acids other than methionine. Mol Cell Biol 18, 5140–5147.PubMedGoogle Scholar
  8. 8.
    C, Kohrer C, Kenny E et al. (2003) Anticodon sequence mutants of Escherichia coli initiator tRNA: Effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation. Biochemistry 42, 4787–4799.Google Scholar
  9. 9.
    Chattapadhyay R, Pelka H, Schulman LH (1990) Initiation of in vivo protein synthesis with non-methionine amino acids. Biochemistry 29, 4263–4268.PubMedCrossRefGoogle Scholar
  10. 10.
    Mamaev S, Olejnik J, Olejnik E K, Rothschild KJ (2004) Cell-free N-terminal protein labeling using initiator suppressor tRNA. Anal Biochem 326, 25–32.PubMedCrossRefGoogle Scholar
  11. 11.
    Olejnik J, Gite S, Mamaev S, Rothschild KJ (2005) N-terminal labeling of proteins using initiator tRNA. Methods 36, 252–260.PubMedCrossRefGoogle Scholar
  12. 12.
    Muranaka N, Miura M, Taira H, Hohsaka T (2007) Incorporation of unnatural non-α-amino acids into the N terminus of proteins in a cell-free translation system. ChemBioChem 8, 1650–1653.PubMedCrossRefGoogle Scholar
  13. 13.
    Miura M, Muranaka N, Abe R, Hohsaka T (2010) Incorporation of fluorescent-labeled non-α-amino carboxylic acids into the N-terminus of proteins in response to amber initiation codon. Bull Chem Soc Jpn 83, 546–553.CrossRefGoogle Scholar
  14. 14.
    Robertson S A, Ellman J A, Schultz P G (1991) A general and efficient route for chemical aminoacylation of transfer RNAs. J Am Chem Soc 113, 2722–2729.CrossRefGoogle Scholar
  15. 15.
    Thorson JS, Cornish VW, Barrett J E et al. (1998) A biosynthetic approach for the incorporation of unnatural amino acids into proteins. Methods Mol Biol 77, 43–73.PubMedGoogle Scholar
  16. 16.
    Baklanov MM, Golikova LN, Malygin E G (1996) Effect on DNA transcription of nucleotide sequences upstream to T7 promoter. Nucleic Acids Res 24, 3659–3660.PubMedCrossRefGoogle Scholar
  17. 17.
    Kao C, Zheng M, Rudisser S (1999) A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5, 1268–1272.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.School of Materials ScienceJapan Advanced Institute of Science and TechnologyNomiJapan

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