Figure 3

N-glycan structure on recombinant secreted human DNase I. a: Band-shift assay: rhDNase I from CHO cells and secreted rhDNase I from T. thermophila were de-glycosylated. The treatment with PNGase F (F+) results in a significant band-shift, when compared to the controls (F-), illustrating that the secreted rhDNase I of T. thermophila becomes glycosylated. As T. thermophila rhDNase I had been concentrated by immunoprecipitation, additional bands of antibody-chains appear in Western blotting. The DNase signals are highlighted by asterisks. b: Concanavalin A pulldown assay: Supernatants of transformed T. thermophila strain and a wildtype strain (B1868.7) were analysed. The supernatant of the wildtype was spiked with equal amounts (2 μg) of glycosylated (F-) or de-glycosylated (F+) rhDNase I from CHO cells, respectively. Only mannose-terminated glycosylated rhDNase is recovered by Con A (lane 3), glycosylated (F-) and de-glycosylated rhDNase I (F+) from CHO cells did not bind to ConA beads (lanes 1 and 2). c: DSA-FACE N-glycan analysis of T. thermophila rhDNase I: This data illustrates the highly consistent N-glycan structure on rhDNaseI produced in T. thermophila. All N-glycans are mannose terminated, as shown by digestion with jack bean mannosidase in the lower panel. The most prominent structure is the Man₃GlcNAc₂ core pattern with additional alpha 1,2 bound mannoses (two middle panels). d: DSA-FACE N-glycan analysis of CHO rhDNase I: On the CHO derived rhDNase I more than 20 distinct peaks, each corresponding to a different N-glycan, are observed in the untreated sample. This typical complex profile of mammalian cells is in sharp contrast to the consistent T. thermophila glycosylation. Even removal of terminal N-acetylneuraminic acid by treatment with sialidase still leads to a large number of various glycans (lower panel).