Expression of functional human DNaseI. a: The structure concept of the expression cassettes. For all expression cassettes we used the cell-cycle dependent H4-1 histone or the Cd-inducible MTT1 promotor and the beta tubulin 2 (BTU2) terminator sequence. Selection was done by the neo resistance gene (neoR). Secretion of the rhDNase was regulated by parts of the endogenous precursor of PLA1. We used the first 36, 110 and 115 amino acids of the PLA-precursor. The mature human DNase I corresponds to the aa 23–281. b: We obtained an anti DNaseI serum by using CHO derived DNase I (Pulmozyme) as an antigen. After that the serum was tested by western blot using de-glycosylated DNaseI (PNGaseF treated, F+ lane1) as well as the glycosylated protein. No significant difference in the signal strength could be observed, suggesting that the N-glycan structure of rhDNase has no, or only a minor influence on the avidity of the serum. c: Secretion of processed recombinant human DNaseI into the media. Lane1: 20 μl supernatant of the T. thermophila wildtype strain B1868.7, negative control; Lane2: 20 μl supernatant of mock-(EGFP) transformed T. thermophila; Lane 3–5: 20 μl supernatant of T. thermophila cells that were transformed with expression plasmids, carrying the spPLA36, ppPLA110 and ppPLA115 prepro-peptides, respectively. d: Determination of DNase activity in 10 μl supernatant of transformed T. thermophila. Control: mock-(EGFP) expressing cells; Histone: cell cycle dependent expression of ppPLA115; MTT1: inducible expression of ppPLA115; (MTT1-Cd: non-induced; MTT1 + Cd: induced). e: Different amounts of rhDNaseI from CHO cells (1.0, 2.5 and 5.0 ng) were used to roughly quantify the yield of rhDNase in 30 μl ppPLA115 supernatant of T. thermophila. f: Demonstration of up-scalability by comparing cell titres during 50 litre fermentation (black) and 400 ml shaker flask cultivation (gray) of rhDNase I secreting T. thermophila.