Skip to main content
SpringerLink
Log in

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-13C-glucose and 15N-glutamate as labeled precursors. This study suggests that uniformly 15N,13C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altamirano C, Paredes C, Cairo JJ, Godia F (2000) Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine. Biotechnol Prog 16:69–75

    Article  Google Scholar 

  • Altamirano C, Cairo JJ, Godia F (2001a) Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control. Biotechnol Bioeng 76:351–360

    Article  Google Scholar 

  • Altamirano C, Illanes A, Casablancas A, Gamez X, Cairo JJ, Godia C (2001b) Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. Biotechnol Prog 17:1032–1041

    Article  Google Scholar 

  • Altamirano C, Paredes C, Illanes A, Cairo JJ, Godia F (2004) Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J Biotechnol 110:171–179

    Article  Google Scholar 

  • Archer SJ, Bax A, Roberts AB, Sporn MB, Ogwa Y, Piez KA, Weatherbee JA, Tsang ML-S, Lucas J, Zheng B-L, Wenker J, Torchia D (1993) Transforming growth factor βl: NMR signal assignments of the recombinant protein expressed and isotopically enriched using chinese hamster ovary cells. Biochemistry 32:1152–1163

    Article  Google Scholar 

  • Bebbington CR, Hentschel GCG (1987) The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells. In: Glover DM (ed) DNA cloning, vol III. IRL Press, Oxford, pp 163–188

    Google Scholar 

  • Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM (1998) Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 329:191–196

    Google Scholar 

  • Brauman JI (1966) Least squares analysis and simplification of multi-isotope mass spectra. Anal Chem 38:607–610

    Article  Google Scholar 

  • Brüggert M, Rehm T, Shanker S, Georgescu J, Holak TA (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

    Article  Google Scholar 

  • Christie A, Butler M (1999) The adaptation of bhk cells to a non-ammoniagenic glutamate-based culture medium. Biotechnol Bioeng 64:298–309

    Article  Google Scholar 

  • Criscuolo F, Mozo J, Hurtaud C, Nubel T, Bouillaud F (2006) UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism. BBA Bioener 1757:1284–1291

    Article  Google Scholar 

  • Cruz HJ, Moreira JL, Carrondo MJ (2000) Metabolically optimised BHK cell fed-batch cultures. J Biotechnol 80:109–118

    Article  Google Scholar 

  • Curi R, Lagranha CJ, Doi SQ, Sellitti DF, Procopio J, Pithon-Curi TC (2005a) Glutamine-dependent changes in gene expression and protein activity. Cell Struct Funct 23:77–84

    Google Scholar 

  • Curi R, Lagranha CJ, Doi SQ, Sellitti DF, Procopio J, Pithon-Curi TC, Corless M, Newsholme P (2005b) Molecular mechanisms of glutamine action. J Cell Physiol 204:392–401

    Article  Google Scholar 

  • Deshpande R, Yang TH, Heinzle E (2009) Towards a metabolic and isotopic steady state in CHO batch cultures for reliable isotope-based metabolic profiling. Biotechnol J 4:247–263

    Article  Google Scholar 

  • Eagle H (1955) Nutrition needs of mammalian cells in tissue culture. Science 122:501–504

    Article  ADS  Google Scholar 

  • Eagle H (1959) Amino acid metabolism in mammalian cell cultures. Science 130:432–437

    Article  ADS  Google Scholar 

  • Elias CB, Carpentier E, Durocher Y, Bisson L, Wagner R, Kamen A (2003) Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme. Biotechnol Prog 19:90–97

    Article  Google Scholar 

  • Franchi-Gazzola R, Dall’Asta V, Sala R, Visigalli R, Bevilacqua E, Gaccioli F, Gazzola GC, Bussolati O (2006) The role of the neutral amino acid transporter SNAT2 in cell volume regulation. Acta Physiologica 187:273–283

    Article  Google Scholar 

  • Fuchs BC, Bode BP (2006) Stressing out over survival: glutamine as an apoptotic modulator. J Surg Res 131:26–40

    Article  Google Scholar 

  • Fumarola C, Zerbini A, Guidotti GG (2001) Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis. Cell Death Differ 8:1004–1013

    Article  Google Scholar 

  • Fumarola C, La Monica S, Alfieri RR, Borra E, Guidotti GG (2005a) Cell size reduction induced by inhibition of the mTOR/S6 K-signaling pathway protects Jurkat cells from apoptosis. Cell Death Differ 12:1344–1357

    Article  Google Scholar 

  • Fumarola C, La Monica S, Guidotti GG (2005b) Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: role of glutamine and of cell shrinkage. J Cell Physiol 204:155–165

    Article  Google Scholar 

  • Gawlitzek M, Papac DI, Sliwkowski MB, Ryll T (1999) Incorporation of 15 N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells. Glycobiology 9:125–131

    Article  Google Scholar 

  • Goswami J, Sinskey AJ, Steller H, Stephanopoulos GN, Wang DI (1999) Apoptosis in batch cultures of Chinese hamster ovary cells. Biotechnol Bioeng 62:632–640

    Article  Google Scholar 

  • Hansen AP, Petros AM, Mazar AP, Pederson TM, Rueter A, Fesik SW (1992) A practical method for uniform isotope labeling of recombinant proteins in mammalian cells. Biochemistry 31:12713–12718

    Article  Google Scholar 

  • Irani N, Wirth M, van Den Heuvel J, Wagner R (1999) Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction. Biotechnol Bioeng 66:238–246

    Article  Google Scholar 

  • Jennings ME, Matthews DE (2005) Determination of complex isotopomer patterns in isotopically labeled compounds by mass spectrometry. Anal Chem 77:6435–6444

    Article  Google Scholar 

  • Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  • Lee W-NP, Byerley LO, Bergner EA, Edmond J (1991) Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20:451–458

    Article  Google Scholar 

  • Li L, Mi L, Feng Q, Liu R, Tang H, Xie L, Yu X, Chen Z (2005) Increasing the culture efficiency of hybridoma cells by the use of integrated metabolic control of glucose and glutamine at low levels. Biotechnol Appl Biochem 42:73–80

    Article  Google Scholar 

  • Ljunggren J, Haggstrom L (1994) Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures. Biotechnol Bioeng 44:808–818

    Article  Google Scholar 

  • Lloyd S, Brocks C, Chatham JC (2003) Differential modulation of glucose, lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart. Am J Physiol Heart Circ Physiol 285:H163–H172

    Google Scholar 

  • Lu S, Sun X, Zhang Y (2005) Insight into metabolism of CHO cells at low glucose concentration on the basis of the determination of intracellular metabolites. Process Biochem 40:1917–1921

    Article  Google Scholar 

  • 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 7:295–304

    Article  Google Scholar 

  • Maranga M, Goochee CF (2006) Metabolism of PER.C6TM cells cultivated under fed-batch conditions at low glucose and glutamine levels. Biotechnol Bioeng 94:139–150

    Article  Google Scholar 

  • Martinelle K, Doverskog M, Jacobsson U, Chapman BE, Kuchel PW, Haggstrom L (1998) Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: evidence from 1H/15 N NMR. Biotechnol Bioeng 60:508–517

    Article  Google Scholar 

  • Mates JM, Perez-Gomez C, de Castro IN, Asenjo M, Marquez J (2002) Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death. Int J Biochem Cell B 34:439–458

    Article  Google Scholar 

  • Mawhinney TP, Robinett RSR, Atalay A, Madson MA (1986) Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatogaphjy and mass spectrometry. J Chromatogr 358:231–242

    Article  Google Scholar 

  • Mehndiratta P, Walton WJ, Hare JT, Pulido S, Parthasarathy G, Emmett MR, Marshall AG, Logan TM (2004) Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells. Prot Expr Purif 33:274–287

    Article  Google Scholar 

  • Michelakis ED, Webster L, Mackey JR (2008) Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer. Br J Cancer 99:989–994

    Article  Google Scholar 

  • Mori S, Abeygunawardana C, Johnson MON, van Zijl PCM (1995) Improved sensitivity of HSQC spectra of exchangin protons at short interscan delays using a new fast hsqc (FHSQC) detection scheme that avoids water saturation. J Magn Reson 108B:94–98

    Google Scholar 

  • Newsholme P, Lima MMR, Procopio J, Pithon-Curi TC, Doi SQ, Bazotte RB, Curi R (2003a) Glutamine and glutamate as vital metabolites. Braz J Med Biol Res 36:153–163

    Article  Google Scholar 

  • Newsholme P, Procopio J, Lima MMR, Pithon-Curi TC, Curi R (2003b) Glutamine and glutamate - their central role in cell metabolism and function. Cell Struct Funct 21:1–9

    Google Scholar 

  • Öhman L, Ljunggren J, Häggström L (1995) Induction of a metabolic switch in insect cells by substrate-limited fed batch cultures. Appl Microbiol Biotechnol 43:1006–1013

    Article  Google Scholar 

  • Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson 93:151–170

    Google Scholar 

  • Paquette JC, Guerin PJ, Gauthier ER (2005) Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation. J Cell Physiol 202:912–921

    Article  Google Scholar 

  • Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L (2000) Simultaneous analysis of metabolites in potato tuber by gas chromatography–mass spectrometry. Plant J 23:131–142

    Article  Google Scholar 

  • Schleucher J, Schwendinger M, Sattler M, Schmidt P, Schedletzky O, Glaser SJ, Sorensen OW, Griesinger C (1994) A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J Biomol NMR 4:301–306

    Article  Google Scholar 

  • 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 26:367–372

    Article  Google Scholar 

  • 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 31:343–349

    Article  Google Scholar 

  • Street JC, Delort A-M, Braddock PSH, Brindle KM (1993) A 1H/15N n.m.r. study of nitrogen metabolism in cultured mammalian cells. Biochem J 291:485–492

    Google Scholar 

  • Tzatsos A, Tsichlis PN (2007) Energy depletion inhibits phosphatidylinositol 3-kinase/akt signaling and induces apoptosis via AMP-activated protein kinase-dependent phosphorylation of IRS-1 at Ser-794. J Biol Chem 282:18069–18082

    Article  Google Scholar 

  • Van Winden WA, Wittmann C, Heinzle E, Heijnen JJ (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479

    Article  Google Scholar 

  • Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033

    Article  ADS  Google Scholar 

  • Walton WJ, Kasprzak AJ, Hare JT, Logan TM (2006) An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells. J Biomol NMR 36:225–233

    Article  Google Scholar 

  • Ward LD, Matthews JM, Zhang J-G, Simpson RJ (1995) Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates. Biochemistry 34:11652–11659

    Article  Google Scholar 

  • Wittmann C, Hans M, Heinzle E (2002) In vivo analysis of intracellular amino acid labelings by GC/MS. Anal Biochem 307:379–382

    Article  Google Scholar 

  • Wyss DF, Withka JM, Knopeers MH, Sterne KA, Recny MA, Wagner G (1993) 1H resonance assignments and secondary structure of the 13.6 kDa glycosylated adhesion domain of human CD2. Biochemistry 32:10995–11006

    Article  Google Scholar 

  • Wyss DF, Choi JS, Wagner G (1995) Composition and sequence specific resonance assignments of the heterogneous N-linked glycan in the 13.6 kDa ahesion domain fo human CD2 as determined by NMR on the intact glycoprotein. Biochemistry 34:1622–1634

    Article  Google Scholar 

  • Yang M, Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol Bioeng 68:370–380

    Article  Google Scholar 

  • Zanghi JA, Fussenegger M, Bailey JE (1999) Serum protects protein-free competent Chinese hamster ovary cells against apoptosis induced by nutrient deprivation in batch culture. Biotechnol Bioeng 64:108–119

    Article  Google Scholar 

  • Zielke HR, Ozand PT, Tildon JT, Sevdalian DA, Cornblath M (1978) Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J Cell Physiol 95:41–48

    Article  Google Scholar 

Download references

Acknowledgments

TML acknowledges partial support for this research from the FSU Research Foundation, the NMR Program at the National High Magnetic Field Laboratory, and from the NIH (AI21628). This research benefitted from activities at the Southeast Collaboratory for High-Field Biomolecular NMR, a research resource at the University of Georgia, funded by the National Institute of General Medical Sciences (NIGMS grant number GM66340) and the Georgia Research Alliance.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306, USA

    David Skelton, Abbey Goodyear & Timothy M. Logan

  2. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA

    DaQun Ni, Wendy J. Walton, Myron Rolle, Joan T. Hare & Timothy M. Logan

  3. National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, 32310, USA

    Timothy M. Logan

Authors
  1. David Skelton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abbey Goodyear
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. DaQun Ni
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wendy J. Walton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Myron Rolle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joan T. Hare
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Timothy M. Logan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Timothy M. Logan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(DOCX 13455 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Skelton, D., Goodyear, A., Ni, D. et al. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells. J Biomol NMR 48, 93–102 (2010). https://doi.org/10.1007/s10858-010-9440-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-010-9440-x

Keywords

Search

Navigation