Preparation method forEscherichia coli S30 extracts completely dependent upon tRNA addition to catalyze cell-free protein synthesis

  • Jin-Ho Ahn
  • Mi-Yeon Hwang
  • In-Seok Oh
  • Kyung-Moon Park
  • Geun-Hee Hahn
  • Cha-Yong Choi
  • Dong-Myung Kim


A simple method for depletingE. coli S30 extracts of endogenous tRNA has been developed. An ethanolamine-Sepharose® column equilibrated with water selectively captured the tRNA molecules inE. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful forin vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.


cell-free protein synthesis unnatural amino acid in vitro suppression tRNA chloramphenicol acetyltransferase erythropoietin 


  1. [1]
    Noren, C. J., S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz (1989) A general method for site-specific incorporation of unnatural amino acids into proteins.Science 244: 182–188.CrossRefGoogle Scholar
  2. [2]
    Cook, S. N., W. E. Jack, X. Xiong, L. E. Danley, J. A. Ellman, P. G. Schultz, and C. J. Noren (1995) Photochemically initiated protein splicing.Angew. Chem. Int. Edit. 34: 1629–1630.CrossRefGoogle Scholar
  3. [3]
    Miller, J. C., S. K. Silverman, P. M. England, D. A. Dougherty, and H. A. Lester (1998) Flash decaging of tyrosine sidechains in an ion channel.Neuron 20: 619–624.CrossRefGoogle Scholar
  4. [4]
    England, P. M., H. A. Lester, N. Davidson, and D. A. Dougherty (1997) Site-specific, photochemical proteolysis applied to ion channelsin vivo.Proc. Natl. Acad. Sci. USA 94: 11025–11030.CrossRefGoogle Scholar
  5. [5]
    Pollitt, S. K., and P. G. Schultz (1998) A phorochemical switch for controlling protein-protein interactions.Angew. Chem. Int. Edit. 37: 2104–2107.CrossRefGoogle Scholar
  6. [6]
    Cohen, B. E., T. B. McAnaney, E. S. Park, Y. N. Jan, S. G. Boxer, and L. Y. Jan (2002) Probing protein electrostatics with a synthetic fluorescent amino acid.Science 296: 1700–1703.CrossRefGoogle Scholar
  7. [7]
    Steward, L. E., C. S. Collins, M. A. Gilmore, J. E. Carlson, J. B. A. Ross, and A. R. Chamberlin (1997)In vitro site-specific incorporation of fluorescent probes into beta-galactosidase.J. Am. Chem. Soc. 119: 6–11.CrossRefGoogle Scholar
  8. [8]
    Turcatti, G., K. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel, and A. Chollet (1996) Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites.J. Biol. Chem. 271: 19991–19998.CrossRefGoogle Scholar
  9. [9]
    Taki, M., T. Hohsaka, H. Murakami, K. Taira, and M. Sisido (2002) Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs.J. Am. Chem. Soc. 124: 14586–14590.CrossRefGoogle Scholar
  10. [10]
    Cornish, V. W., D. R. Benson, C. A. Altenbach, K. Hideg, W. L. Hubbell, and P. G. Schultz (1994) Site-specific incorporation of biophysical probes into proteins.Proc. Natl. Acad. Sci. USA 91: 2910–2914.CrossRefGoogle Scholar
  11. [11]
    Cornish, V. W., K. M. Hahn, and P. G. Schultz (1996) Site-specific protein modification using a ketone handle.J. Am. Chem. Soc. 118: 8150–8151.CrossRefGoogle Scholar
  12. [12]
    Wang, L., Z. W. Zhang, A. Brock, and P. G. Schultz (2003) Addition of the keto functional group to the genetic code ofEscherichia coli.Proc. Natl. Acad. Sci. USA 100: 56–61.CrossRefGoogle Scholar
  13. [13]
    Wang, L., A. Brock, B. Herberich, and P. G. Schultz (2001) Expanding the genetic code ofEscherichia coli.Science 292: 498–500.CrossRefGoogle Scholar
  14. [14]
    Chin, J. W., T. A. Cropp, J. C. Anderson, M. Mukherji, Z. W. Zhang, and P. G. Schultz (2003) An expanded eukaryotic genetic code.Science 301: 964–967.CrossRefGoogle Scholar
  15. [15]
    Taira, H., T. Hohsaka, and M. Sisido (2006)in vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in anEscherichia coli cell-free translation system.Nucleic Acids Res. 34: 1653–1662.CrossRefGoogle Scholar
  16. [16]
    Hohsaka, T., Y. Ashizuka, H. Murakami, and M. Sisido (2001) Five-base codons for incorporation of nonnatural amino acids into proteins.Nucleic Acids Res. 29: 3646–3651.CrossRefGoogle Scholar
  17. [17]
    Kanda, T., K. Takai, T. Hohsaka, M. Sisido, and H. Takaku (2000) Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein.Biochem. Biophys. Res. Commun. 270: 1136–1139.CrossRefGoogle Scholar
  18. [18]
    Hohsaka, T., Y. Ashizuka, H. Taira, H. Murakami, and M. Sisido (2001) Incorporation of nonnatural amino acids into proteins by using various four-base codons in anEscherichia coli in vitro translation system.Biochemistry 40: 11060–11064.CrossRefGoogle Scholar
  19. [19]
    Anderson, J. C., T. J. Magliery, and P. G. Schultz (2002) Exploring the limits of codon and anticodon size.Chem. Biol. 9: 237–244.CrossRefGoogle Scholar
  20. [20]
    Anderson, J. C., N. Wu, S. W. Santoro, V. Lakshman, D. S. King, and P. G. Schultz (2004) An expanded genetic code with a functional quadruplet codon.Proc. Natl. Acad. Sci. USA 101: 7566–7571.CrossRefGoogle Scholar
  21. [21]
    Frankel, A., and R. W. Roberts (2003)In vitro selection for sense codon suppression.RNA 9: 780–786.CrossRefGoogle Scholar
  22. [22]
    Jackson, R. J., S. Napthine, and I. Brierley (2001) Development of a tRNA-dependentin vitro translation system.RNA 7: 765–773.CrossRefGoogle Scholar
  23. [23]
    Son, J. M., J. H. Ahn, M. Y. Hwang, C. G. Park, C. Y. Choi, and D. M. Kim (2006) Enhancing the efficiency of cell-free protein synthesis through the polymerase-chainreaction-based addition of a translation enhancer sequence and thein situ removal of the extra amino acid residues.Anal. Biochem. 351: 187–192.CrossRefGoogle Scholar
  24. [24]
    Kim, D. M., T. Kigawa, C. Y. Choi, and S. Yokoyama (1996) A highly efficient cell-free protein synthesis system fromEscherichia coli.Eur. J. Biochem. 239: 881–886.CrossRefGoogle Scholar
  25. [25]
    Nakano, H., R. Okumura, C. Goto, and T. Yamane (2002)In vitro combinatorial mutagenesis of the 65th and 222nd positions of the green fluorescent protein ofAequarea victoria.Biotechnol. Bioprocess Eng. 7: 311–315.CrossRefGoogle Scholar
  26. [26]
    Lee, C.-S., S.-H. Lee, Y.-G. Kim, C.-H. Choi, Y.-K. Kim, and B.-G. Kim (2006) Biochemical reactions on a micro-fluidic chip based on a precise fluidic handling method at the nanoliter scale.Biotechnol. Bioprocess Eng. 11: 146–153.CrossRefGoogle Scholar
  27. [27]
    Park, Y. S., S. H. Hwang, and C.-Y. Choi (2005) Construction of CpG motif-enriched DNA vaccine plasmids for enhanced early immune response.Biotechnol. Bioprocess Eng. 10: 29–33.CrossRefGoogle Scholar
  28. [28]
    Kim, D.-M., C.-Y. Choi, J.-H. Ahn, T.-W. Kim, N.-Y. Kim, I.-S. Oh, and C.-G. Park (2006) Development of a rapid and productive cell-free protein synthesis system.Biotechnol. Bioprocess Eng. 11: 235–239.CrossRefGoogle Scholar
  29. [29]
    Schagger, H., and G. von Jagow (1987) Trieine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.Anal. Biochem. 166: 368–379.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 2006

Authors and Affiliations

  • Jin-Ho Ahn
    • 1
  • Mi-Yeon Hwang
    • 2
  • In-Seok Oh
    • 2
  • Kyung-Moon Park
    • 3
  • Geun-Hee Hahn
    • 4
  • Cha-Yong Choi
    • 1
    • 2
  • Dong-Myung Kim
    • 4
  1. 1.Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of EngineeringSeoul National UniversitySeoulKorea
  2. 2.School of Chemical and Biological Engineering, College of EngineeringSeoul National UniversitySeoulKorea
  3. 3.Department of Chemical System EngineeringHongik UniversityChungnamKorea
  4. 4.Department of Fine Chemical Engineering and ChemistryChungnam National UniversityDaejeonKorea

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