Extraction and purification of decorin from corneal stroma retain structure and biological activity.
We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44kDa protein identified as decorin with a yield of 35mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [125I]TGF-beta1 with an apparent K(d) of 40nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.
Christopher T
CT
Brown
P
P
Lin
Mary T
MT
Walsh
Donald
D
Gantz
Matthew A
MA
Nugent
V
V
Trinkaus-Randall
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003094:Collagen / Q000096:biosynthesis; D003319:Corneal Stroma / Q000737:chemistry*; D058575:Decorin; D016326:Extracellular Matrix Proteins; D013058:Mass Spectrometry; D011489:Protein Denaturation; D011509:Proteoglycans / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism
D058575:Decorin; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; C028326:concanavalin A-sepharose; C037405:heparin-sepharose; D003094:Collagen; D012685:Sepharose