Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase
Figure 6
Glycosylation analysis of recombinant GCR secreted from high-producer subclone (SC 12.9) by western blotting assays. (a) Endoglycosidase digestion with Endo H and PNGase F. Samples of conditioned medium (supernatant of 150,000 cells corresponding to about 750 ng of GCR) were precipitated with 10% TCA previously to digestion reactions. Non-digested sample of nontransfected CHO-DXB11 cells was used as negative control. Nonglycosylated recombinant GCR purified from E. coli was a positive control. Glycosylated GCR bands of 66–69 kDa and nonglycosylated GCR of 56 kDa are indicated by solid and dashed arrows, respectively. (b) Purified commercial enzyme Cerezyme (150 ng) of 60 kDa containing remodeled glycosylation moieties was compared to GCR secreted from subclone 12.9 (supernatant of 30,000 cells corresponding to about 150 ng) with complex- and hybrid-type glycosylation pattern (66–69 kDa).
