Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells.
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.
Skelton
David
D
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
Goodyear
Abbey
A
Ni
Daqun
D
Walton
Wendy J
WJ
Rolle
Myron
M
Hare
Joan T
JT
Logan
Timothy M
TM
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D003999:Dichloroacetic Acid / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D007553:Isotope Labeling / Q000379:methods*; D019344:Lactic Acid / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000596:Amino Acids; D002247:Carbon Isotopes; D006023:Glycoproteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D019344:Lactic Acid; D003999:Dichloroacetic Acid; D005947:Glucose