Fan H: Expression, purification, and characterization of human dipeptidyl peptidase

The humandipeptidyl peptidase IV/CD26 (DPPIV/CD26) is amultifunctional type-IImembrane bound glycoprotein.As a receptor of collagen I and fibronectin it mediates cell–cell and cell–matrix adhesion, and by interacting with extracellular adenosine deaminase and CD45 it is involved in regulatory and costimulatory events in the immune system. DPPIV/CD26 has a very distinct substrate specificity, and is potentially capable of truncating many cytokines, chemokines, and peptide hormones. In this study, we describe the overexpression, purification, and characterization of human DPPIV/CD26 in Spodoptera frugiperda (Sf9) cells, using the baculovirus system. Overexpression of DPPIV/CD26 was confirmed by measurement of its peptidase specificity, SDS–PAGE, and Western blot analyses. Expression rates were between 6.4 and 17.6mg protein per liter suspension culture (1:5 109 cells). The N-linked oligosac-charide composition was examined and compared with that of mammalian cell-expressed DPPIV/CD26. Two-step purification by immunoaffinity chromatography and size-exclusion fast protein liquid chromatography (SE-FPLC) led to highly stable protein with significant peptidase activity. A subsequent gel filtration step on a Superdex 200 column yielded 2mg homogeneous dimeric DPPIV/ CD26 (per liter insect cell culture) for crystallographic studies. Protein homogeneity was confirmed by silver staining of non-denatu