Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

  • Hideo TakahashiEmail author
  • Ichio ShimadaEmail author


The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.


Stable isotope-labeling Prokaryotic cell Eukaryotic cell Heterologous proteins 



The authors thank Drs. Noritaka Nishida, Toshihiko Sugiki, and Osamu Ichikawa for useful discussions. This work was financially supported in part by the Ministry of Economy, Trade and Industry (METI) and the New Energy and Industrial Technology Development Organization (NEDO).


  1. Arata Y, Kato K, Takahashi H, Shimada I (1994) Nuclear magnetic resonance study of antibodies: a multinuclear approach. Methods Enzymol 239:440–464CrossRefGoogle Scholar
  2. Brüggert M, Rehm T, Shanker S, Georgescu J, Holak TA (2003) A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells. J Biomol NMR 25:335–348CrossRefGoogle Scholar
  3. Chen CY, Cheng CH, Chen YC, Lee JC, Chou SH, Huang W, Chuang WJ (2006) Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris. Proteins 62:279–287CrossRefGoogle Scholar
  4. Colussi PA, Taron CH (2005) Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis. Appl Environ Microbiol 71:7092–7098CrossRefGoogle Scholar
  5. Creemers AF, Klaassen CH, Bovee-Geurts PH, Kelle R, Kragl U, Raap J, de Grip WJ, Lugtenburg J, de Groot HJ (1999) Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin. Biochemistry 38:7195–7199CrossRefGoogle Scholar
  6. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323Google Scholar
  7. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52CrossRefGoogle Scholar
  8. Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358CrossRefGoogle Scholar
  9. Gardner KH, Zhang X, Gehring K, Kay LE (1998) Solution NMR studies of 42 kDa Escherichia coli maltose binding protein/β-cyclodextrin complex: chemical shift assignments and analysis. J Am Chem Soc 120:11738–11748CrossRefGoogle Scholar
  10. Georgiou G, Segatori L (2005) Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr Opin Biotechnol 16:538–545CrossRefGoogle Scholar
  11. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5:135–146CrossRefGoogle Scholar
  12. Hansen AP, Petros AM, Mazar AP, Pederson TM, Rueter A, Fesik SW (1992) A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells. Biochemistry 31:12713–12718CrossRefGoogle Scholar
  13. Ichikawa O, Osawa M, Nishida N, Goshima N, Nomura N, Shimada I (2007) Structural basis of the collagen-binding mode of discoidin domain receptor 2. EMBO J 26:4168–4176CrossRefGoogle Scholar
  14. Kay LE, Gardner KH (1997) Solution NMR spectroscopy beyond 25 kDa. Curr Opin Struct Biol 7:722–731CrossRefGoogle Scholar
  15. Klein-Seetharaman J, Reeves PJ, Loewen MC, Getmanova EV, Chung J, Schwalbe H, Wright PE, Khorana HG (2002) Solution NMR spectroscopy of [α-15N] lysine-labeled rhodopsin: the single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence. Proc Natl Acad Sci U S A 99:3452–3457CrossRefADSGoogle Scholar
  16. Klein-Seetharaman J, Yanamala NV, Javeed F, Reeves PJ, Getmanova EV, Loewen MC, Schwalbe H, Khorana HG (2004) Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR. Proc Natl Acad Sci U S A 101:3409–3413CrossRefADSGoogle Scholar
  17. Laroche Y, Storme V, De Meutter J, Messens J, Lauwereys M (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Biotechnology (N Y) 12:1119–1124CrossRefGoogle Scholar
  18. Lundstrom K (2006) Structural genomics for membrane proteins. Cell Mol Life Sci 63:2597–2607CrossRefGoogle Scholar
  19. Lundstrom K, Wagner R, Reinhart C, Desmyter A, Cherouati N, Magnin T, Zeder-Lutz G, Courtot M, Prual C, Andre N, Hassaine G, Michel H, Cambillau C, Pattus F (2006) Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. J Struct Funct Genomics 7:77–91CrossRefGoogle Scholar
  20. Lustbader JW, Birken S, Pollak S, Pound A, Chait BT, Mirza UA, Ramnarain S, Canfield RE, Brown JM (1996) Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures. J Biomol NMR 7:295–304CrossRefGoogle Scholar
  21. Massou S, Puech V, Talmont F, Demange P, Lindley ND, Tropis M, Milon A (1999) Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts. J Biomol NMR 14:231–239CrossRefGoogle Scholar
  22. Merico A, Capitanio D, Vigentini I, Ranzi BM, Compagno C (2004) How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis. J Biotechnol 109:139–146CrossRefGoogle Scholar
  23. Morgan WD, Kragt A, Feeney J (2000) Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies. J Biomol NMR 17:337–347CrossRefGoogle Scholar
  24. Nakanishi T, Miyazawa M, Sakakura M, Terasawa H, Takahashi H, Shimada I (2002) Determination of the interface of a large protein complex by transferred cross-saturation measurements. J Mol Biol 318:245–249CrossRefGoogle Scholar
  25. Nisius L, Rogowski M, Vangelista L, Grzesiek S (2008) Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES. Protein Expr Purif 61:155–162CrossRefGoogle Scholar
  26. O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors. Oxford University Press, New YorkGoogle Scholar
  27. Ohki S, Dohi K, Tamai A, Takeuchi M, Mori M (2008) Stable-isotope labeling using an inducible viral infection system in suspension-cultured plant cells. J Biomol NMR 42:271–277CrossRefGoogle Scholar
  28. Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22:877–882CrossRefGoogle Scholar
  29. Rodriguez E, Krishna NR (2001) An economical method for 15N/13C isotopic labeling of proteins expressed in Pichia pastoris. J Biochem 130:19–22Google Scholar
  30. Ruan KH, Cervantes V, Wu J (2008) A simple, quick, and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies. Biochemistry 47:6819–6826CrossRefGoogle Scholar
  31. Schein CH, Noteborn MHM (1988) Formation of soluble recombinant proteins in Escherichia colli is favored by lower growth temperature. Biotechnology 6:291–294CrossRefGoogle Scholar
  32. Shinagawa M, Shimba N, Mizukoshi T, Arashida N, Yamada N, Kikuchi Y, Suzuki E (2005) High expression with Corynebacterium glutamicum for nuclear magnetic resonance sample preparation. Anal Biochem 344:281–283Google Scholar
  33. Shindo K, Masuda K, Takahashi H, Arata Y, Shimada I (2000) Backbone 1H, 13C, and 15N resonance assignments of the anti-dansyl antibody Fv fragment. J Biomol NMR 17:357–358CrossRefGoogle Scholar
  34. Strauss A, Bitsch F, Cutting B, Fendrich G, Graff P, Liebetanz J, Zurini M, Jahnke W (2003) Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies. J Biomol NMR 26:367–372CrossRefGoogle Scholar
  35. Strauss A, Bitsch F, Fendrich G, Graff P, Knecht R, Meyhack B, Jahnke W (2005) Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J Biomol NMR 31:343–349CrossRefGoogle Scholar
  36. Sugiki T, Shimada I, Takahashi H (2008) Stable isotope labeling of protein by Kluyveromyces lactis for NMR study. J Biomol NMR 42:159–162CrossRefGoogle Scholar
  37. Takahashi H, Nakanishi T, Kami K, Arata Y, Shimada I (2000) A novel NMR method for determining the interfaces of large protein–protein complexes. Nat Struct Biol 7:220–223CrossRefGoogle Scholar
  38. Tanio M, Tanaka T, Kohno T (2008) 15N isotope labeling of a protein secreted by Brevibacillus choshinensis for NMR study. Anal Biochem 373:164–166CrossRefGoogle Scholar
  39. Tanio M, Tanaka R, Tanaka T, Kohno T (2009) Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis. Anal Biochem 386:156–160CrossRefGoogle Scholar
  40. Tomida M, Kimura M, Kuwata K, Hayashi T, Okano Y, Era S (2003) Development of a high-level expression system for deuterium-labeled human serum albumin. Jpn J Physiol 53:65–69CrossRefGoogle Scholar
  41. Udaka S, Yamagata H (1993) High-level secretion of heterologous proteins by Bacillus brevis. Methods Enzymol 217:23–33CrossRefGoogle Scholar
  42. Vajpai N, Strauss A, Fendrich G, Cowan-Jacob SW, Manley PW, Grzesiek S, Jahnke W (2008) Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib. J Biol Chem 283:18292–18302CrossRefGoogle Scholar
  43. Vogt TC, Schinzel S, Bechinger B (2003) Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis. J Biomol NMR 26:1–11CrossRefGoogle Scholar
  44. Werner K, Richter C, Klein-Seetharaman J, Schwalbe H (2008) Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy. J Biomol NMR 40:49–53zbMATHCrossRefGoogle Scholar
  45. Wood MJ, Komives EA (1999) Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation. J Biomol NMR 13:149–159CrossRefGoogle Scholar
  46. Yin J, Li G, Ren X, Herrler G (2007) Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 127:335–347CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Biomedicinal Information Research Center (BIRC)National Institute of Advanced Industrial Science and Technology (AIST)TokyoJapan
  2. 2.Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan

Personalised recommendations