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Cell-Free Protein Synthesis Using E. coli Cell Extract for NMR Studies

  • Mitsuhiro Takeda
  • Masatsune KainoshoEmail author
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 992)

Abstract

The use of cell-free protein production systems for producing isotope labeled proteins generates new opportunities to perform unprecedented NMR studies. As compared with conventional cellular expression systems, the scrambling and dilution of amino acids are highly suppressed in the cell-free reaction, allowing the production of proteins with a wide variety of residue and site-specific isotope labeling patterns. In this chapter, the procedure for cell-free protein synthesis for NMR studies, using an E. coli extract, is introduced.

Keywords

Dialysis Solution Label Amino Acid Protein Synthesis Machinery Endogenous Amino Acid Isotope Label Amino Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Kigawa T, Muto Y, Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J Biomol NMR 6:129–134PubMedCrossRefGoogle Scholar
  2. 2.
    Zubay G (1973) In vitro synthesis of protein in microbial systems. Ann Rev Genet 7:267–287PubMedCrossRefGoogle Scholar
  3. 3.
    Spirin AS, Baranov VI, Ryabova LA, Ovodov SY, Alakhov YB (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242:1162–1164PubMedCrossRefGoogle Scholar
  4. 4.
    Kim DM, Kigawa T, Choi CY, Yokoyama S (1996) A highly efficient cell-free protein synthesis system from Escherichia coli. Eur J Biochem 239:881–886PubMedCrossRefGoogle Scholar
  5. 5.
    Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T, Yokoyama S (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 442:15–19PubMedCrossRefGoogle Scholar
  6. 6.
    Kramer G, Kudlicki W, Hardesty B (1999) Cell-free coupled transcription-translation systems from Escherichia coli. Oxford University Press, New York, pp 129–165Google Scholar
  7. 7.
    Kim DM, Swartz JR (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotechnol Prog 16:385–390PubMedCrossRefGoogle Scholar
  8. 8.
    Madin K, Sawasaki T, Ogasawara T, Endo Y (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc Natl Acad Sci USA 97:559–564PubMedCrossRefGoogle Scholar
  9. 9.
    Endo Y, Sawasaki T (2003) High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. Biotechnol Adv 21:695–713PubMedCrossRefGoogle Scholar
  10. 10.
    Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755PubMedCrossRefGoogle Scholar
  11. 11.
    Shimizu Y, Ueda T (2010) PURE technology. Methods Mol Biol 607:11–21PubMedCrossRefGoogle Scholar
  12. 12.
    Parker MJ, Aulton-Jones M, Hounslow AM, Craven CJ (2004) A combinatorial selective labeling method for the assignment of backbone amide NMR resonances. J Am Chem Soc 126:5020–5021PubMedCrossRefGoogle Scholar
  13. 13.
    Wu PS, Ozawa K, Jergic S, Su XC, Dixon NE, Otting G (2006) Amino-acid type identification in 15N-HSQC spectra by combinatorial selective 15N-labelling. J Biomol NMR 34:13–21PubMedCrossRefGoogle Scholar
  14. 14.
    Ozawa K, Headlam MJ, Mouradov D, Watt SJ, Beck JL, Rodgers KJ, Dean RT, Huber T, Otting G, Dixon NE (2005) Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins. FEBS J 272:3162–3171PubMedCrossRefGoogle Scholar
  15. 15.
    Ugwumba IN, Ozawa K, de la Cruz L, Xu ZQ, Herlt AJ, Hadler KS, Coppin C, Brown SE, Schenk G, Oakeshott JG, Otting G (2011) Using a genetically encoded fluorescent amino acid as a site-specific probe to detect binding of low-molecular-weight compounds. Assay Drug Dev Technol 9:50–57PubMedCrossRefGoogle Scholar
  16. 16.
    Loscha KV, Herlt AJ, Qi R, Huber T, Ozawa K, Otting G (2012) Multiple-site labeling of proteins with unnatural amino acids. Angew Chem Int Ed 51:1–5CrossRefGoogle Scholar
  17. 17.
    Sobhanifar S, Reckel S, Junge F, Schwarz D, Kai L, Karbyshev M, Löhr F, Bernhard F, Dötsch V (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J Biomol NMR 46:33–43PubMedCrossRefGoogle Scholar
  18. 18.
    Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dötsch V, Bernhard F (2011) Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins. N Biotechnol 28:262–271PubMedCrossRefGoogle Scholar
  19. 19.
    Reckel S, Gottstein D, Stehle J, Löhr F, Verhoefen MK, Takeda M, Silvers R, Kainosho M, Glaubitz C, Wachtveitl J, Bernhard F, Schwalbe H, Güntert P, Dötsch V (2011) Solution NMR structure of proteorhodopsin. Angew Chem Int Ed Engl 50:11942–11946PubMedCrossRefGoogle Scholar
  20. 20.
    Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Ono AM, Güntert P (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57PubMedCrossRefGoogle Scholar
  21. 21.
    Kainosho M, Güntert P (2009) SAIL-Stereo-array isotope labeling. Q Rev Biophys 7:1–54Google Scholar
  22. 22.
    Takeda M, Chang CK, Ikeya T, Güntert P, Chang YH, Hsu YL, Huang TH, Kainosho M (2008) Solution structure of the C-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method. J Mol Biol 380:608–622PubMedCrossRefGoogle Scholar
  23. 23.
    Takeda M, Sugimori N, Torizawa T, Terauchi T, Ono AM, Yagi H, Yamaguchi Y, Kato K, Ikeya T, Jee J, Güntert P, Aceti DJ, Markley JL, Kainosho M (2008) Structure of the putative 32 kDa myrosinase binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR. FEBS J 275:5873–5884PubMedCrossRefGoogle Scholar
  24. 24.
    Takeda M, Jee J, Ono AM, Terauchi T, Kainosho M (2011) Hydrogen exchange study on the hydroxyl groups of serine and threonine residues in proteins and structure refinement using NOE restraints with polar side-chain groups. J Am Chem Soc 133:17420–17427PubMedCrossRefGoogle Scholar
  25. 25.
    Kim DM, Choi CY (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol Prog 12:645–649PubMedCrossRefGoogle Scholar
  26. 26.
    Ozawa K, Headlam MJ, Schaeffer PM, Henderson BR, Dixon NE, Otting G (2004) Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy. Eur J Biochem 271:4084–4093PubMedCrossRefGoogle Scholar
  27. 27.
    Torizawa T, Shimizu M, Taoka M, Miyano H, Kainosho M (2004) Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol. J Biomol NMR 30:311–325PubMedCrossRefGoogle Scholar
  28. 28.
    Nirenberg M (1963) Cell-free protein synthesis directed by messenger RNA. Methods Enzymol 6:17–23CrossRefGoogle Scholar
  29. 29.
    Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. In: Hames BD, Higgins SJ (eds) Transcription and translation: a practical approach. IRL Press, Oxford, pp 179–209Google Scholar
  30. 30.
    Liu DV, Zawada JF, Swartz JR (2005) Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis. Biotechnol Prog 21:460–465PubMedCrossRefGoogle Scholar
  31. 31.
    Takeda M, Ikeya T, Güntert P, Kainosho M (2007) Automated structure determination of proteins with the SAIL-FLYA NMR method. Nat Protoc 2:2896–2902PubMedCrossRefGoogle Scholar
  32. 32.
    Takeda M, Kainosho M (2012) Cell-free protein production for NMR studies. Methods Mol Biol 831:71–84PubMedCrossRefGoogle Scholar
  33. 33.
    Schindler PT, Baumann S, Reuss M, Siemann M (2000) In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity. Electrophoresis 21:2606–2609PubMedCrossRefGoogle Scholar
  34. 34.
    Zawada J, Swartz JR (2006) Effects of growth rate on cell extract performance in cell-free protein synthesis. Biotechnol Bioeng 94:618–624PubMedCrossRefGoogle Scholar
  35. 35.
    Kim TW, Keum JW, Oh IS, Choi CY, Park CG, Kim DM (2006) Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system. J Biotechnol 126:554–561PubMedCrossRefGoogle Scholar
  36. 36.
    Pedersen A, Hellberg K, Enberg J, Karlsson BG (2011) Rational improvement of cell-free protein synthesis. N Biotechnol 30:218–224CrossRefGoogle Scholar
  37. 37.
    Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89PubMedCrossRefGoogle Scholar
  38. 38.
    Wu PS, Ozawa K, Lim SP, Vasudevan SG, Dixon NE, Otting G (2007) Cell-free transcription/translation from PCR-amplified DNA for high-throughput NMR studies. Angew Chem Int Ed Engl 46:3356–3358PubMedCrossRefGoogle Scholar
  39. 39.
    Kawarasaki Y, Nakano H, Yamane T (1998) Phosphatase-immunodepleted cell-free protein synthesis system. J Biotechnol 61:199–208PubMedCrossRefGoogle Scholar
  40. 40.
    Etezady-Esfarjani T, Hiller S, Villalba C, Wüthrich K (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39:229–238PubMedCrossRefGoogle Scholar
  41. 41.
    Tonelli M, Singarapu KK, Makino S, Sahu SC, Matsubara Y, Endo Y, Kainosho M, Markley JL (2011) Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition. J Biomol NMR 51:467–476PubMedCrossRefGoogle Scholar
  42. 42.
    Jia X, Ozawa K, Loscha K, Otting G (2009) Glutarate and N-acetyl-L-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins. J Biomol NMR 44:59–67PubMedCrossRefGoogle Scholar
  43. 43.
    Yokoyama J, Matsuda T, Koshiba S, Tochio N, Kigawa T (2011) A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution. Anal Biochem 411:223–229PubMedCrossRefGoogle Scholar
  44. 44.
    Su XC, Loh CT, Qi R, Otting G (2011) Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O. J Biomol NMR 50:35–42PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  1. 1.Structural Biology Research Center, Graduate School of ScienceNagoya UniversityNagoyaJapan
  2. 2.Center for Priority Areas, Graduate School of Science and TechnologyTokyo Metropolitan UniversityHachiojiJapan

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