An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells

Abstract

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.

Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system

“…Metabolic isotope-labeling of glycoproteins by non-mammalian cells has been reported for NMR analyses, e.g. Thy-1 expressed by Sf9 cells [55], glycosylated fragment of thrombomodulin by Pichia pastorisis [56], and a soluble form of prespore-specific antigen by Dictyostelium discoideum [57]. However, it should be noted that glycosylation heavily depends on the host species [53,54].…”

Isotope Labeling of Proteins in Insect Cells

“…Uniform 13 C or 15 N, 13 C labeling can be performed using commercial Bioexpress 2000 (CIL; Hamatsu et al, 2013;Strauss et al, 2005) or homemade media (Sitarska et al, 2015;Walton, Kasprzak, Hare, & Logan, 2006). For carbon labeling, algal extracts are the most economic source of amino acids, as they can be produced from 13 CO 2 instead of more complex and more expensive carbohydrates.…”

Isotope Labeling in Insect Cells

“…Two approaches have been followed: Preparing the media from scratch [ 1 ] or ordering amino acid-free versions of commercial media, i.e. without amino acids and yeast extract [ 2,5,13,18 ] .…”

“…On top of this, the yeast extract as well as the carbohydrates need to be isotope labeled. There are two records of uniform labeling in insect cells, one using selfmade medium [ 18 ] and one relying on a commercial medium [ 14 ] . In the fi rst approach, a 15 N labeled algal extract was used as the main amino acid source (1.5 g/l).…”