Expression, Purification, and Kinetic Characterization of Full-Length Human Fibroblast Activation Protein

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The kcat is 2.0 s−1 and kcat/Km is 1.0 × 104 M−1 s−1 at pH 8.5. The pH dependence of kcat reveals two ionization groups with pKa1 of 7.0 and pKa2 of 11.0. The pH profile of kcat/Km yields similar results with pKa1 6.2 and pKa2 11.0. The neutral pKa1 is associated with His at the active site. The basic pKa2 might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.