Fig. 6
From: Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells
NMU and PYY enzyme immunoassays require amidation for full cross-reactivity. a. Characterization of NMU-amide assay using synthetic peptides. Left: the medium from PAM/820 s cells exhibited a dilution pattern parallel to the amidated standard peptide. Synthetic NMU-8-NH2 (Bachem) was almost identical to the full 25-residue NMU-NH2 in the assay (not shown). By contrast, the Gly-extended synthetic peptide and CHO medium from PAM/820 s cells after BCS treatment showed similar flat, partial dilution curves. Right: BCS in a dose-dependent manner inhibited the amidation of NMU by the PAM/820 s cells, using data from 1 to 6 μl of medium. Secretion of Fc-NMU was not affected (not shown). b. Left: Characterization of PYY-amide assay using synthetic peptides. Left: medium from CHO cells expressing PAM1 diluted in parallel with the authentic PYY-NH2 standard, while the Gly-extended peptide and medium from BCS-treated cells exhibited a much flatter dilution profile. Right: 50 μM BCS inhibited amidation of PYY, using data from 0.5 to 2 μl medium. For both NMU and PYY, data from several assays were normalized using their respective standards to enable the comparisons