Fig. 10. Recombinant CMG-2 binds to the basement membrane matrix proteins, collagen
type IV and laminin. The 20 kDa domain proximal (residues 34-214) to the
putative transmembrane domain was expressed as a histidine-tagged fusion
protein in E.coli and purified by Ni/Cd-sepharose chromatography. Five µg
of the protein was run on a 12% SDS-PAGE gel and was stained by Coomassie blue
(A). A schematic diagram of CMG-2 is shown with the von Willebrand Factor A
domain homology noted from residues 43-212 which is within the 20 kDa
recombinantly produced protein (B). A control protein, histidine-tagged GFP,
was similarly purified as a control. These proteins were absorbed to plastic
at 10 µg/ml and after blocking with detergent, were incubated with 1
µg/ml of biotinylated collagen type IV, laminin, osteopontin, fibronectin
and human serum albumin in 0.1% Tween-20 in Tris-buffered saline, pH 7.5.
After a 1 hour incubation at 25°C, the wells were washed and then
incubated with 1 µg/ml of avidin-peroxidase in the same Tween-20-Tris
buffer for 30 minutes. The wells were then washed, developed for peroxidase
activity and the plate read in an ELISA plate reader at 490 nm. The values
shown are derived from triplicate wells + s.d. (C).
