Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells
| Autor: | Abbey Goodyear, Myron Rolle, Joan Hare, DaQun Ni, Timothy M. Logan, David Skelton, Wendy J. Walton |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Pyruvate dehydrogenase kinase
Cell Survival CHO Cells Biochemistry law.invention Cricetulus law Cricetinae Animals Lactic Acid Viability assay Amino Acids Nuclear Magnetic Resonance Biomolecular Spectroscopy Glycoproteins chemistry.chemical_classification Carbon Isotopes Dichloroacetic Acid Nitrogen Isotopes Chinese hamster ovary cell Glutamate receptor Recombinant Proteins Amino acid Glutamine Glucose chemistry Isotope Labeling Recombinant DNA Glycoprotein |
| Zdroj: | Journal of Biomolecular NMR. 48:93-102 |
| ISSN: | 1573-5001 0925-2738 |
| DOI: | 10.1007/s10858-010-9440-x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink