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).