The production and purification of functional decorin in a baculovirus system.
Human decorin was expressed in Spodoptera frugiperda 21 (Sf21) insect cells. A full-length cDNA encoding preprodecorin of 359 amino acids from a human fibroblast library was cloned into baculovirus transfer vector pVL1392, and transfected into Sf21 insect cells. The infected cells secreted the mature decorin into the culture medium. The secreted decorin lacked glycosaminoglycan but was N-glycosylated, whereas the unmodified decorin was present in the cell lysates, suggesting that N-glycosylation is required for decorin secretion from Sf21 cells. The recombinant decorin was then efficiently purified from the conditioned medium by two chromatographic procedures, hydroxyapatite Sepharose and Con A-Agarose, under nondenaturing conditions. The purified decorin was more potent, as evaluated by the inhibition of collagen fibrillogenesis, than that obtained from bovine tissues under denaturing conditions. The final yield of recombinant decorin was 1.5 mg in 200 ml culture medium of 3 x 10(8) cells. The biologically active decorin produced in Sf21 cells is a potentially useful probe for investigating the molecular interactions of this protein with other extracellular matrix proteins and may also have therapeutic applications.
Gu
J
J
Department of Molecular Medicine, Osaka Medical Center, Japan.
Nakayama
Y
Y
Nagai
K
K
Wada
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003094:Collagen / Q000037:antagonists & inhibitors; D018076:DNA, Complementary; D058575:Decorin; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006801:Humans; D011509:Proteoglycans / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
C546878:DCN protein, human; D018076:DNA, Complementary; D058575:Decorin; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D003094:Collagen