Journal of Biomolecular NMR

, Volume 50, Issue 1, pp 35–42 | Cite as

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

  • Xun-Cheng Su
  • Choy-Theng Loh
  • Ruhu Qi
  • Gottfried Otting


Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that reduction of E. coli S30 extracts with NaBH4 presents a simple and inexpensive way to achieve cleaner selective isotope labelling in cell-free protein synthesis reactions. The purpose of the NaBH4 is to inactivate all pyridoxal-phosphate (PLP) dependent enzymes by irreversible reduction of the Schiff bases formed between PLP and lysine side chains of the enzymes or amino groups of free amino acids. The reduced S30 extracts retain their activity of protein synthesis, can be stored as well as conventional S30 extracts and effectively suppress conversions between different amino acids. In addition, inactivation of PLP-dependent enzymes greatly stabilizes hydrogens bound to α-carbons against exchange with water, minimizing the loss of α-deuterons during cell-free production of proteins from perdeuterated amino acids in H2O solution. This allows the production of highly perdeuterated proteins that contain protons at all exchangeable positions, without having to back-exchange labile deuterons for protons as required for proteins that have been synthesized in D2O.


Cell-free protein synthesis Isotope scrambling NaBH4 Pyridoxal phosphate Selective 15N labelling 



We thank Mr. Xinying Jia and Dr. Hiromasa Yagi for help with the NMR spectroscopy and Cambridge Isotope Laboratories for generous gifts of 2H/15N/13C-labelled amino acids and amino acid mixture. Financial support by the Australian Research Council is gratefully acknowledged.


  1. Apponyi MA, Ozawa K, Dixon NE, Otting G (2008) Cell-free protein synthesis for analysis by NMR spectroscopy. In: Kobe B, Guss M, Huber T (eds) Methods in molecular biology 426, structural proteomics: high-throughput methods. Humana Press, Totowa, pp 257–268Google Scholar
  2. Berg JM, Tymoczko JL, Stryer L (2006) Biochemistry. WH Freeman and Company, New YorkGoogle Scholar
  3. Eliot AC, Kirsch JF (2004) Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu Rev Biochem 73:383–4515CrossRefGoogle Scholar
  4. Etezady-Esfarjani T, Hiller S, Villalba C, Wüthrich K (2007) Cell-free synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39:229–238CrossRefGoogle Scholar
  5. Guignard L, Ozawa K, Pursglove SE, Otting G, Dixon NE (2002) NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach. FEBS Lett 524:159–162CrossRefGoogle Scholar
  6. 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 44:59–67CrossRefGoogle Scholar
  7. Kainosho M, Güntert P (2010) SAIL–stereo-array isotope labeling. Q Rev Biophys 42:247–300CrossRefGoogle Scholar
  8. Kariya E, Ohki S, Hayano T, Kainosho M (2000) Backbone 1H, 13C, and 15N resonance assignments of an 18.2 kDa protein, E. coli peptidyl-prolyl cis-trans isomerase b (EPPIb). J Biomol NMR 18:75–76CrossRefGoogle Scholar
  9. Kigawa T, Muto Y, Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable-isotope labelling of proteins for NMR analysis. J Biomol NMR 6:129–134CrossRefGoogle Scholar
  10. LeMaster DM (1990) Deuterium labeling in NMR structural analysis of larger proteins. Quart Rev Biophys 23:133–174CrossRefGoogle Scholar
  11. McIntosh LP, Dahlquist FW (1990) Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear-magnetic resonance spectra of proteins. Quart Rev Biophys 23:1–38CrossRefGoogle Scholar
  12. Michuda CM, Martinez-Carrion M (1970) The isozymes of glutamate-aspartate transaminase. Mechanism of inhibition by dicarboxylic acids. J Biol Chem 245:262–269Google Scholar
  13. Morino Y, Nagashima F (1984) Pyridoxal phosphate-binding site in enzymes–reduction and comparison of sequences. Methods Enzymol 106:116–137CrossRefGoogle Scholar
  14. Morita EH, Shimizu M, Ogasawara T, Endo Y, Tanaka R, Kohno T (2004) A novel way of amino acid-specific assignment in 1H–15N HSQC spectra with a wheat germ cell-free protein synthesis system. J Biomol NMR 30:37–45CrossRefGoogle Scholar
  15. Muchmore DC, McIntosh LP, Russell CB, Anderson DE, Dahlquist FW (1989) Expression and 15N labeling of proteins for proton and 15N nuclear magnetic resonance. Methods Enzymol 177:44–73CrossRefGoogle Scholar
  16. 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 15N-labelled proteins for rapid analysis by NMR spectroscopy. Eur J Biochem 271:4084–4093CrossRefGoogle Scholar
  17. Ozawa K, Wu PSC, Dixon NE, Otting G (2006) 15N-Labelled proteins by cell-free protein synthesis: strategies for high-throughput NMR studies of proteins and protein-ligand complexes. FEBS J 273:4154–4159CrossRefGoogle Scholar
  18. Prusiner S, Stadtman ER (1976) Regulation of glutaminase B in Escherichia coli. II. Modulation of activity by carboxylate and borate ions. J Biol Chem 251:3457–3462Google Scholar
  19. Shi J, Pelton JG, Cho HS, Wemmer DE (2004) Protein signal assignments using specific labeling and cell-free synthesis. J Biomol NMR 28:235–247CrossRefMATHGoogle Scholar
  20. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotech 19:751–755CrossRefGoogle Scholar
  21. Shortle D (1994) Assignment of amino-acid type in 1H–15N correlation spectra by labeling with 14N-amino acids. J Magn Reson B 105:88–90CrossRefGoogle Scholar
  22. 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 46:33–43CrossRefGoogle Scholar
  23. Staunton D, Schlinker R, Zanetti G, Colebrook SA, Campbell ID (2006) Cell-free expression and selective isotope labelling in protein NMR. Magn Reson Chem 44:S2–S9CrossRefGoogle Scholar
  24. Su XC, Jergic S, Keniry MA, Dixon NE, Otting G (2007) Solution structure of domains IVa and V of the τ subunit of Escherichia coli DNA polymerase III and interaction with the α subunit. Nucl Acids Res 35:2813–2824CrossRefGoogle Scholar
  25. Takeuchi K, Ng E, Malia TJ, Wagner G (2007) 1–13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins. J Biomol NMR 38:89–98CrossRefGoogle Scholar
  26. Toney MD (2005) Reaction specificity in pyridoxal phosphate enzymes. Arch Biochem Biophys 433:279–287CrossRefGoogle Scholar
  27. Tong KI, Yamamoto M, Tanaka T (2008) A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli. J Biomol NMR 42:59–67CrossRefGoogle Scholar
  28. Waugh DS (1996) Genetic tools for selective labeling of proteins with α-15N-amino acids. J Biomol NMR 8:184–192CrossRefGoogle Scholar
  29. Weisbrod RE, Meister A (1973) Studies on glutamine synthetase from Escherichia coli: formation of pyrrolidone carboxylate and inhibition by methionine sulfoximine. J Biol Chem 248:3997–4002Google Scholar
  30. Wu PSC, 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 34:13–21CrossRefGoogle Scholar
  31. Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y, Laue ED, Cooper JA, Kainosho M, Yokoyama S (1998) Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR 11:295–306CrossRefGoogle Scholar
  32. Yokoyama J, Matsuda T, Koshiba S, Kigawa T (2010) An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system. J Biomol NMR 48:193–201CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Xun-Cheng Su
    • 1
    • 2
  • Choy-Theng Loh
    • 1
  • Ruhu Qi
    • 1
  • Gottfried Otting
    • 1
  1. 1.Research School of ChemistryAustralian National UniversityCanberraAustralia
  2. 2.State Key Laboratory of Elemento-Organic ChemistryNankai UniversityTianjinChina

Personalised recommendations