Purification of decorin core protein from human lung tissue.
A chromatographic method to purify decorin core protein from human lung tissue is described. The method is simple and rapid, using a combination of two-anion exchange and one reversed phase chromatography steps and the enzymatic digestion with chondroitinase ABC. Approximately 170 microg decorin core protein were purified from 25 g of lung tissue with an enrichment factor of 1800-fold relative to the initial protein content. SDS-PAGE analysis of the final product revealed a single 42 kDa protein band, which was recognized by anti-decorin antibodies upon Western blotting and identified by mass spectrometry. Further digestion with PNGase F evidenced the presence of three N-linked oligosaccharides on the core protein. This method forms the basis for studying structural alterations of decorin related to the pathology of diseases where tissue destruction plays a role.
Didraga
Mihaela
M
University Center for Pharmacy, Department of Analytical Biochemistry, 9700 AD, Groningen, The Netherlands.
Barroso
Begona
B
de Vries
Marcel
M
Kerstjens
Huib
H
Postma
Dirkje
D
Bischoff
Rainer
R
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002845:Chromatography / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D058575:Decorin; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism; D012907:Smoking; D014357:Trypsin / Q000378:metabolism
C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D014357:Trypsin