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,
glypicans 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.