Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the expression, cellular presentation, function and analysis of the soluble human hetIL-15 complex. a. Engineered human IL-15 and IL-15Rα were co-expressed and secreted by HEK293 cells as a soluble heterodimeric complex (hetIL-15) after proteolytic cleavage from the cell surface. The complex binds to the IL-2Rβ/γ receptor complex located on target cells, where it initiates a cellular response. b. The hetIL-15 complex was isolated and its purity monitored using reducing (upper) and native (lower) SDS-PAGE with Coomassie blue staining (left gels) and Western blotting using anti-IL-15 and anti-IL-15Rα antibodies (right gels). The two clinically relevant preparations of hetIL-15 (i.e. the EN and cGMP lots) are shown. c. IL-15 and sIL-15Rα were separated from their heterodimeric complex by non-reductive RP-HPLC and individually subjected to LC-MS/MS-based glycan (top), glycopeptide (bottom) and glycoprotein (right) profiling in order to characterize their N- and O-glycosylation in a detailed and site-specific manner

Article Snippet: Slightly different conditions were used to separate the two examined hetIL-15 preparations ( i.e. , EN and cGMP lots): The EN lot of hetIL-15 was separated at a flow rate of 5 ml/min on a preparative PLRP-S polymeric C18 RP-HPLC column (i.d., 50 mm, length, 150 mm, Agilent).

Techniques: Expressing, Isolation, SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy