ADAMTS5, Recombinant, His-Tag (Aggrecanase 2 A Disintegrin And Metalloproteinase with ThromboSpondin-3 motif)

Aggrecanases are glutamyl-specific multidomain metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family. They hydrolyze aggrecan, the major proteoglycan of articular cartilage, causing osteoarthritis and other joint diseases. Recent studies using knockout mice have indicated that ADAMTS-5 (aggrecanase-2) plays an essential role in aggrecan degradation in mice. Produced with the baculo-virus expression system and purified from insect cell culture supernatants. The protein contains the catalytic domain, the disintegrin domain and the thrombospondin type-1 motif of full-length ADAMTS-5 followed by a C-terminal His6-tag. The calculated Mr of the amino acid sequence is 40.8kD. In SDS-PAGE the protein exhibits a Mr of about 50kD. Applications: Suitable to be used to study the degradation of extracellular matrix proteoglycans, to screen for inhibitors of proteoglycan hydrolysis and to characterize inhibitor actions. The enzyme can also serve as standard in enzymatic and immunological assays. Activity: Aggrecanase activity of the ADAMTS-5 preparation is determined with recombinant aggrecan interglobular domain (Aggrecan-IGD). ADAMTS-5 hydrolyzes the "aggrecanase" site within this domain (peptide bond E373 -A374 in human aggrecan). The recombinant substrate is incubated at a concentration of 0.1uM with ADAMTS-5 in 50mM Tris-HCl, pH 7.5, 150mM sodium chloride, 5mM CaCl2, 1uM leupeptin, 1uM pepstatin, mM Pefabloc, 0.05 % Brij 35 for 15 min at 37 degrees C. Substrate cleavage at the "aggrecanase"-site is estimated from the appearance of the hydrolysis fragment with the novel N-terminus ARGSVIL. The fragment is quantified with two monoclonal antibodies. Under the specified conditions the hydrolysis rate is > 0.5nmoles hydrolyzed substrate/min/ml ADAMTS-5 preparation or > 5nmoles hydrolyzed substrate/min/mg. Inhibitors: ADAMTS 5 is inhibited by tissue inhibitor of matrix metalloproteinase 3 (TIMP3) and by alpha2-macroglobulin. Enzyme activity is also suppressed by chelators of divalent cations such as EDTA and by synthetic metalloproteinase inhibitors. Epitope: Recombinant human truncated Storage and Stability: Aliquot to avoid repeated freezing and thawing and freeze at -70 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 12 months.