Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase
Figure 1
Strategy of cloning of GCR cDNA in the mammalian expression vector pED. (a) Map of dicistronic vector pED, a pUC 18 derivative, containing the simian virus 40 (SV40) origin of replication and enhancer element, the adenovirus major late promoter (AdMLP), the tripartite leader from adenovirus late mRNA (AdTPL), a hybrid intron composed of the 5′ splice site from the first leader of adenovirus major late mRNAs and a 3′ splice site from an immunoglobulin gene (IVS), a multiple cloning site (SMC), the 5′ untranslated leader from encephalomyocarditis (EMC-L) virus, a murine DHFR-coding region, the simian virus 40 late polyadenylation signal (SV40-pA), and ampicillin resistance gene for propagation and selection in E. coli () [26]. GCR cDNA was cloned into XbaI and EcoRI sites. (b) Amino acid sequence of cloned GCR cDNA. The signal peptide of 19 amino acids is underlined. Potential carbohydrate binding asparagine residues are shown by diamonds. The arginine at position 495 was replaced by histidine (mutation R495H) (boxed).
