An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells.
It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with 15N and carbohydrates with 13C by NMR spectroscopy.
Walton
Wendy J
WJ
Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
Kasprzak
Agnieszka J
AJ
Hare
Joan T
JT
Logan
Timothy M
TM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002247:Carbon Isotopes / Q000737:chemistry*; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media / Q000737:chemistry / Q000191:economics*; D005947:Glucose / Q000032:analysis; D005973:Glutamine / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D018800:Thy-1 Antigens / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D002247:Carbon Isotopes; D003470:Culture Media; D006023:Glycoproteins; D009587:Nitrogen Isotopes; D011506:Proteins; D018800:Thy-1 Antigens; D005973:Glutamine; D005947:Glucose