Figure 2

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.