Expression, purification, and characterization of human dipeptidyl peptidase IV/CD26 in Sf9 insect cells.
The human dipeptidyl peptidase IV/CD26 (DPPIV/CD26) is a multifunctional type-II membrane 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 17.6 mg protein per liter suspension culture (1.5 x 10(9)cells). The N-linked oligosaccharide 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-denaturating PAGE gels and by MALDI-TOF analysis of tryptic peptides.
Institut für Molekularbiologie und Biochemie, Fachbereich Humanmedizin, Freie Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
Jörg
J
Dobers
Martin
M
Zimmermann-Kordmann
Melanie
M
Leddermann
Tina
T
Schewe
Werner
W
Reutter
Hua
H
Fan
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D037102:Lectins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*
D018076:DNA, Complementary; D037102:Lectins; D011994:Recombinant Proteins; D018819:Dipeptidyl Peptidase 4