Journal of Biomolecular NMR

, Volume 62, Issue 2, pp 191–197 | Cite as

Affordable uniform isotope labeling with 2H, 13C and 15N in insect cells

  • Agnieszka Sitarska
  • Lukasz Skora
  • Julia Klopp
  • Susan Roest
  • César Fernández
  • Binesh Shrestha
  • Alvar D. GossertEmail author


For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for 15N and 13C with yields comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.


Eukaryotic cells Insect cells Protein expression Isotope labeling Uniform isotope labeling NMR Deuteration 



We would like to thank Alexandra Hinniger for help with purification of protein and Jan Hendrik Rieger and Chrystèle Henry for help with sample preparation.

Conflict of interest

The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10858_2015_9935_MOESM1_ESM.pdf (972 kb)
Supplementary material 1 (PDF 971 kb)


  1. Brüggert M, Rehm T, Shanker S et al (2003) A novel medium for expression of proteins selectively labeled with 15 N-amino acids in Spodoptera frugiperda (Sf9) insect cells. J Biomol NMR 25:335–348. doi: 10.1023/A:1023062906448 CrossRefGoogle Scholar
  2. Dong S, Wang M, Qiu Z et al (2010) Autographa californica multicapsid nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH. J Virol 84:5351–5359. doi: 10.1128/JVI.02517-09 CrossRefGoogle Scholar
  3. Doverskog M, Han L, Häggström L (1998) Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth. Cytotechnology 26:91–102. doi: 10.1023/A:1007963003607 CrossRefGoogle Scholar
  4. Egorova-Zachernyuk TA, Bosman GJCGM, Pistorius AMA, DeGrip WJ (2009) Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture. Appl Microbiol Biotechnol 84:575–581. doi: 10.1007/s00253-009-2063-z CrossRefGoogle Scholar
  5. Gossert AD, Jahnke W (2012) Isotope labeling in insect cells. In: Atreya HS (ed) Isotope labeling in biomolecular NMR. Springer, Dordrecht, pp 179–196CrossRefGoogle Scholar
  6. Gossert AD, Hinniger A, Gutmann S et al (2011) A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility. J Biomol NMR 51:449–456. doi: 10.1007/s10858-011-9570-9 CrossRefGoogle Scholar
  7. Hansen AP, Petros AM, Mazar AP et al (1992) A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells. Biochemistry (Mosc) 31:12713–12718. doi: 10.1021/bi00166a001 CrossRefGoogle Scholar
  8. Hefferon KL, Oomens AGP, Monsma SA et al (1999) Host cell receptor binding by baculovirus GP64 and kinetics of virion entry. Virology 258:455–468. doi: 10.1006/viro.1999.9758 CrossRefGoogle Scholar
  9. Kofuku Y, Ueda T, Okude J et al (2014) Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy. Angew Chem Int Ed 53:13376–13379. doi: 10.1002/anie.201406603 CrossRefGoogle Scholar
  10. Li M, Smith CJ, Walker MT, Smith TJ (2009) Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics. J Biol Chem 284:22988–23000. doi: 10.1074/jbc.M109.020222 CrossRefGoogle Scholar
  11. Linser R, Gelev V, Hagn F et al (2014) Selective methyl labeling of eukaryotic membrane proteins using cell-free expression. J Am Chem Soc 136:11308–11310. doi: 10.1021/ja504791j CrossRefGoogle Scholar
  12. Meola A, Deville C, Jeffers SA et al (2014) Robust and low cost uniform 15 N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications. J Struct Biol 188:71–78. doi: 10.1016/j.jsb.2014.08.002 CrossRefGoogle Scholar
  13. O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus expression vectors: a laboratory manual. W.H. Freeman and Co, New YorkGoogle Scholar
  14. Öhman L, Alarcon M, Ljunggren J et al (1996) Glutamine is not an essential amino acid for Sf-9 insect cells. Biotechnol Lett 18:765–770. doi: 10.1007/BF00127885 CrossRefGoogle Scholar
  15. Rej R (1977) Aminooxyacetate is not an adequate differential inhibitor of aspartate aminotransferase isoenzymes. Clin Chem 23:1508–1509Google Scholar
  16. Strauss A, Bitsch F, Cutting B et al (2003) Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies. J Biomol NMR 26:367–372. doi: 10.1023/A:1024013111478 CrossRefGoogle Scholar
  17. Strauss A, Bitsch F, Fendrich G et al (2005) Efficient uniform isotope labeling of Abl kinase expressed in baculovirus-infected insect cells. J Biomol NMR 31:343–349. doi: 10.1007/s10858-005-2451-3 CrossRefGoogle Scholar
  18. Takahashi H, Shimada I (2009) Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells. J Biomol NMR 46:3–10. doi: 10.1007/s10858-009-9377-0 CrossRefGoogle Scholar
  19. Vajpai N, Strauss A, Fendrich G et al (2008) Backbone NMR resonance assignment of the Abelson kinase domain in complex with imatinib. Biomol NMR Assign 2:41–42. doi: 10.1007/s12104-008-9079-7 CrossRefGoogle Scholar
  20. Vaughn JL, Goodwin RH, Tompkins GJ, McCawley P (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13:213–217. doi: 10.1007/BF02615077 CrossRefGoogle Scholar
  21. Wong DT, Fuller RW, Molloy BB (1973) Inhibition of amino acid transaminases by l-cycloserine. Adv Enzyme Regul 11:139–154CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Novartis Institutes for BioMedical ResearchBaselSwitzerland

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