Figure 2

Characterization of recombinant full-length hiAP. A: Western blot analysis of cell extracts. In a positive clone hiAP expression was induced by the addition of cadmium to the medium. Extracts from induced cells showed a hiAP signal in the Western blot. Extracts from wild type cells (induced and non-induced) and the non-induced cells of the positive clone showed no signal. An extract from transformed CHO cells served as positive control. The double band probably corresponds to intracellular precursor hiAP with N-terminal signal peptide or to a non-cleaved GPI anchor signal. B: The data shown in the Western blot were confirmed by an alkaline phosphatase activity assay. The samples derived from wild type cells treated with and without cadmium (wt +Cd; wt -Cd) and extracts from the non-induced hiAP clone (+GPI -Cd) showed only basal activity. In contrast to this an elevated enzyme activity could be observed in cell extracts from hiAP expressing cells (+GPI +Cd), suggesting that recombinant hiAP is expressed as an active enzyme. C: We treated extracts of the hiAP expressing cells with the enzyme PNgase F (F+). Extracts from CHO cells expressing hiAP were used as a positive control. Negative controls were non-treated cell extracts (F-). The results show a significant band shift, indicating that both CHO derived as well as T. thermophila derived hiAP carries N-glycans. As expected, the band shift in ciliates is less significant due to the smaller N-glycan structure. In contrast to mammalian proteins that carry a complex N-glycosylation T. thermophila has most probably an N-glycan structure of the oligo-mannose Man₃GlcNAc₂ type (see scheme).