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Unread 08-14-2010, 04:19 AM
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Default Enhanced production and isotope enrichment of recombinant glycoproteins produced in c

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.
  • Content Type Journal Article
  • DOI 10.1007/s10858-010-9440-x
  • Authors
    • David Skelton, Florida State University Department of Chemistry and Biochemistry Tallahassee FL 32306 USA
    • Abbey Goodyear, Florida State University Department of Chemistry and Biochemistry Tallahassee FL 32306 USA
    • DaQun Ni, Florida State University Institute of Molecular Biophysics Tallahassee FL 32306 USA
    • Wendy J. Walton, Florida State University Institute of Molecular Biophysics Tallahassee FL 32306 USA
    • Myron Rolle, Florida State University Institute of Molecular Biophysics Tallahassee FL 32306 USA
    • Joan T. Hare, Florida State University Institute of Molecular Biophysics Tallahassee FL 32306 USA
    • Timothy M. Logan, Florida State University Department of Chemistry and Biochemistry Tallahassee FL 32306 USA

Source: Journal of Biomolecular NMR
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