Journal of Biomolecular NMR

, Volume 71, Issue 4, pp 263–273 | Cite as

Improving yields of deuterated, methyl labeled protein by growing in H2O

  • Evan S. O’Brien
  • Danny W. Lin
  • Brian Fuglestad
  • Matthew A. Stetz
  • Travis Gosse
  • Cecilia Tommos
  • A. Joshua Wand


Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15N–1H TROSY and 13C–1H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2O leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in H2O, with the addition of 2H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in H2O M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in D2O M9 for a variety of protein targets while still achieving 75–80% deuteration. Because the procedure does not require bulk D2O or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific 13CH3 labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of 2H/15N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.


Protein deuteration Nuclear magnetic resonance Amino acid chromatography ILVM methyl labeling Algal amino acids 



We thank L. Liang for assistance throughout this project. We thank Professor Trevor M. Penning for the AKR1c3 expression vector. This work was supported by the G. Harold and Leila Y. Mathers Foundation and NIH Grant GM079190. E.S.O. is a NIH predoctoral trainee (T32 GM008275).

Author contributions

E.S.O., D.W.L. & A.J.W. designed the research. E.S.O., D.W.L., M.A.S., B.F., & T.G. prepared samples and carried out NMR experiments. C.T. assisted in method implementation. E.S.O. and A.J.W. wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.


  1. Cai M, Huang Y, Sakaguchi K, Clore GM, Gronenborn AM, Craigie R (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J Biomol NMR 11:97–102CrossRefGoogle Scholar
  2. Cai M, Huang Y, Yang R, Craigie R, Clore GM (2016) A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks. J Biomol NMR 66:85–91CrossRefGoogle Scholar
  3. Caro JA et al (2017) Entropy in molecular recognition by proteins. Proc Natl Acad Sci USA 114:6563–6568CrossRefGoogle Scholar
  4. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  5. Desmond JC et al (2003) The aldo-keto reductase AKR1C3 is a novel suppressor of cell differentiation that provides a plausible target for the non-cyclooxygenase-dependent antineoplastic actions of nonsteroidal anti-inflammatory drugs. Cancer Res 63:505–512Google Scholar
  6. Englander SW (2006) Hydrogen exchange and mass spectrometry: A historical perspective. J Am Soc Mass Spectrom 17:1481–1489CrossRefGoogle Scholar
  7. Etezady-Esfarjani T, Hiller S, Villalba C, Wuthrich K (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39:229–238CrossRefGoogle Scholar
  8. Fiaux J, Bertelsen EB, Horwich AL, Wuthrich K (2002) NMR analysis of a 900K GroEL GroES complex. Nature 418:207–211ADSCrossRefGoogle Scholar
  9. Fiaux J, Bertelsen EB, Horwich AL, Wuthrich K (2004) Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background. J Biomol NMR 29:289–297CrossRefGoogle Scholar
  10. Fike R (2009) Nutrient supplementation strategies for biopharmaceutical production Part 2. Bioprocess Tech 7(11):46–52Google Scholar
  11. Freundlich M, Burns RO, Umbarger HE (1962) Control of isoleucine, valine, and leucine biosynthesis. I. Multivalent repression. Proc Natl Acad Sci USA 48:1804–1808ADSCrossRefGoogle Scholar
  12. Gautier A, Nietlispach D (2012) Solution NMR studies of integral polytopic alpha-helical membrane proteins: the structure determination of the seven-helix transmembrane receptor sensory rhodopsin II, pSRII. Methods Mol Biol 914:25–45Google Scholar
  13. Gautier A, Kirkpatrick JP, Nietlispach D (2008) Solution-state NMR spectroscopy of a seven-helix transmembrane protein receptor: backbone assignment, secondary structure, and dynamics. Angew Chem Int Ed Engl 47:7297–7300CrossRefGoogle Scholar
  14. Gautier A, Mott HR, Bostock MJ, Kirkpatrick JP, Nietlispach D (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol 17:768–774CrossRefGoogle Scholar
  15. Giovanni RD (1960) The effects of deuterium oxide on certain microorganisms. Ann NY Acad Sci 84:644–647ADSCrossRefGoogle Scholar
  16. Goddard TD, Kneller DG (2008) SPARKY 3. University of California, San FranciscoGoogle Scholar
  17. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR 13:369–374CrossRefGoogle Scholar
  18. Grzesiek S, Bax A (1992) Improved 3D triple resonance NMR techniques applied to a 31 kDa protein. J Magn Reson 96:432–440ADSGoogle Scholar
  19. Hohenfeld IP, Wegener AA, Engelhard M (1999) Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli. FEBS Lett 442:198–202CrossRefGoogle Scholar
  20. Hyberts SG, Milbradt AG, Wagner AB, Arthanari H, Wagner G (2012) Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. J Biomol NMR 52:315–327CrossRefGoogle Scholar
  21. Jeschke G (2012) DEER distance measurements on proteins. Annu Rev Phys Chem 63:419–446ADSCrossRefGoogle Scholar
  22. Kay LE, Ikura M, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514ADSGoogle Scholar
  23. Linser R, Gelev V, Hagn F, Arthanari H, Hyberts SG, Wagner G (2014) Selective methyl labeling of eukaryotic membrane proteins using cell-free expression. J Am Chem Soc 136:11308–11310CrossRefGoogle Scholar
  24. Liu W, Flynn PF, Fuentes EJ, Kranz JK, McCormick M, Wand AJ (2001) Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena. Biochemistry 40:14744–14753CrossRefGoogle Scholar
  25. Mironova OS, Efremov RG, Person B, Heberle J, Budyak IL, Buldt G, Schlesinger R (2005) Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli. FEBS Lett 579:3147–3151CrossRefGoogle Scholar
  26. Neylon C (2008) Small angle neutron and X-ray scattering in structural biology: recent examples from the literature. Eur Biophys J 37:531–541CrossRefGoogle Scholar
  27. Ollerenshaw JE, Tugarinov V, Kay LE (2003) Methyl TROSY: explanation and experimental verification. Magn Reson Chem 41:843–852CrossRefGoogle Scholar
  28. Penning TM (2017) Aldo-keto reductase regulation by the Nrf2 system: implications for stress response, chemotherapy drug resistance, and carcinogenesis. Chem Res Toxicol 30:162–176CrossRefGoogle Scholar
  29. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar
  30. Rance M, Loria JP, Palmer AG (1999) Sensitivity improvement of transverse relaxation-optimized spectroscopy. J Magn Reson 136:92–101ADSCrossRefGoogle Scholar
  31. Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98–102ADSCrossRefGoogle Scholar
  32. Ruschak AM, Kay LE (2010) Methyl groups as probes of supra-molecular structure, dynamics and function. J Biomol NMR 46:75–87CrossRefGoogle Scholar
  33. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622CrossRefGoogle Scholar
  34. Sprangers R, Li X, Mao X, Rubinstein JL, Schimmer AD, Kay LE (2008) TROSY-based NMR evidence for a novel class of 20S proteasome inhibitors. Biochemistry 47:6727–6734CrossRefGoogle Scholar
  35. Stetz MA, Carter MV, Wand AJ (2016) Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain. Protein Expr Purif 123:75–82CrossRefGoogle Scholar
  36. Sun H, Kay LE, Tugarinov V (2011) An optimized relaxation-based coherence transfer NMR experiment for the measurement of side-chain order in methyl-protonated, highly deuterated proteins. J Phys Chem B 115:14878–14884CrossRefGoogle Scholar
  37. Tripp JA, McCullagh JS (2012) Preparative HPLC separation of underivatized amino acids for isotopic analysis. Methods Mol Biol 828:339–350CrossRefGoogle Scholar
  38. Tzeng SR, Pai MT, Kalodimos CG (2012) NMR studies of large protein systems. Methods Mol Biol 831:133–140CrossRefGoogle Scholar
  39. Wand AJ, Urbauer JL, McEvoy RP, Bieber RJ (1996) Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein. Biochemistry 35:6116–6125CrossRefGoogle Scholar
  40. Zhang X, Chien EY, Chalmers MJ, Pascal BD, Gatchalian J, Stevens RC, Griffin PR (2010) Dynamics of the beta2-adrenergic G-protein coupled receptor revealed by hydrogen-deuterium exchange. Anal Chem 82:1100–1108CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Johnson Research Foundation and Department of Biochemistry & BiophysicsUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaUSA
  2. 2.Department of Biochemistry & BiophysicsUniversity of PennsylvaniaPhiladelphiaUSA

Personalised recommendations