FIG. 3.

GP-2ecto forms stable trimers and adopts an entirely α-helical structure. GP-2ecto was expressed in E. coli and purified from inclusion bodies by Ni-nitrilotriacetic acid affinity chromatography. The urea-denatured protein was refolded by rapid dilution, and reconstituted GP-2ecto complexes were separated by Superdex 75 gel filtration chromatography. (A) Glutaraldehyde cross-linking analysis of the gel filtration-purified GP-2ecto complexes. Cross-linking was carried out for 10 min at room temperature. Monomeric GP-2ecto has a theoretical molecular mass of 15 kDa. Molecular mass markers (in kDa) are noted at the left of blots. (B) Circular dichroism spectrum for the GP-2ecto complexes, which was recorded in PBS, pH 7.0, at 4°C immediately after gel filtration purification.