Journal of Biomolecular NMR

, Volume 48, Issue 2, pp 93–102 | Cite as

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

  • David Skelton
  • Abbey Goodyear
  • DaQun Ni
  • Wendy J. Walton
  • Myron Rolle
  • Joan T. Hare
  • Timothy M. Logan
Article
First Online:
Received:
Accepted:

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.

Keywords

Recombinant glycoprotein Isotope enrichment Mammalian cells NMR Thy1 
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Notes

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.

Supplementary material

10858_2010_9440_MOESM1_ESM.docx (13.1 mb)
(DOCX 13455 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • David Skelton
    • 1
  • Abbey Goodyear
    • 1
  • DaQun Ni
    • 2
  • Wendy J. Walton
    • 2
  • Myron Rolle
    • 2
  • Joan T. Hare
    • 2
  • Timothy M. Logan
    • 1
    • 2
    • 3
  1. 1.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA
  2. 2.Institute of Molecular BiophysicsFlorida State UniversityTallahasseeUSA
  3. 3.National High Magnetic Field LaboratoryFlorida State UniversityTallahasseeUSA

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