An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells
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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.
Keywordsbaculovirus glycoproteins isotopic labeling NMR Spodoptera frugiperda (Sf9) cells Thy-1
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We thank Margaret Seavy and Bruce Smith for their assistance with amino acid and sugar analysis, Dr. Marc Freeman for use of his vapor pressure osmometer, and Jacqueline Kitchen for help in preparing the baculovirus. We also thank John Glushka at the Complex Carbohydrate Research Center (Athens, GA) for collecting spectra. Support for this project was given to T.M.L. from the Florida State University Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, and a Research Foundation grant (015789). This research benefited from facilities at the Southeast Collaboratory for High-Field Biomolecular NMR, a resource funded by the National Institute of General Medical Sciences (GM66340) and the Georgia Research Alliance. W.J.W. was supported by an NSF GK-12 teaching fellowship (DGE-0139299).