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Figure 1
Overview of the construction of a glypican-AP fusion plasmid.  In its native state (A), glypican consists of an extracellular domain attached to the cell-surface through a glycosylphosphotidyl (GPI) anchor (not shown).  From its DNA sequence data (notably the presence of 14 cysteine residues) and anomalously fast migration on non-reducing gels, glypican’s extracellular domain is thought to consist of a compact globular core and a short linear segment that contains varying number of glycosaminoglycan (GAG) attachment sites near the C-terminus. In the fusion protein (B), the GPI anchor is removed and replaced with heat-stable alkaline phosphatase.  To generate the fusion plasmid (C), the appropriate glypican DNA sequence (nucleotides 1 to 1641) is inserted into APtag-2 immediately upstream of the AP-coding sequence.  APtag-2 contains an SV40 origin of replication that selectively enhances the production of the plasmid in certain mammalian cell lines as well as a CMV promoter that promotes the expression of the plasmid.   The expressed fusion protein, lacking a GPI anchor, is secreted into the extracellular medium and can be conveniently harvested.

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