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 k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.
Sun
Shaoxian
S
Applied Biotechnology, The Dupont Pharmaceuticals Company, Experimental Station, Wilmington, Delaware 19880-0336, USA. shaoxian@yahoo.com
Albright
Charles F
CF
Fish
Barbara H
BH
George
Henry J
HJ
Selling
Bernard H
BH
Hollis
Gregory F
GF
Wynn
Richard
R
D000818:Animals; D000951:Antigens, Neoplasm; D016367:Baculoviridae; D014408:Biomarkers, Tumor; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D010450:Endopeptidases; D018093:Gelatinases; D006031:Glycosylation; D006133:Growth Substances / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000099167:Fibroblast Activation Protein Alpha
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D010450:Endopeptidases; D018093:Gelatinases; D006133:Growth Substances; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha