ADAMTS5 Human Recombinant Protein, truncated, His-tagged
Recombinant human ADAMTS5 625-930 is produced with the baculovirus expression system and enriched from insect cell culture supernatants. The protein contains the catalytic domain, the disintegrin domain and the thrombospondin type-1 motif of full-lenth ADAMTS5 followed by a C-terminal His6-tag. The calculated Mr of the amino acid sequence is 40.8 kDa. In SDSPAGE the protein exhibits a Mr of about 50 kDa. The higher Mr is probably due to glycosylation. ADAMTS5 is solublized in 150 mM NaCl, 5 mM CaCl2, 50 mM imidazol, 50 mM Tris-HCl, pH 7.5, 0.05 % Brij-35.
ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) are multidomain extracellular proteinases, containing at least one thrombospondin type-1 repeat . The enzymes are involved in processing of extracellular matrix proteins and proteins in the circulation. ADAMTS5 was first purified from bovine nasal cartilage conditioned media and human ADAMTS5 cDNA was cloned from a human liver cDNA library . ADAMTS5 consists of a prodomain, a catalytic domain, a disintegrin domain, a thrombospondin type-1 motif, a Cys-rich region followed by a spacer sequence and thrombospondin submotifs. The prodomain is most likely cleaved-off by a furin-type enzyme before ADAMTS5 is released from cells.
ADAMTS5 is expressed constitutively in synovium  and it degrades aggrecan, the major proteoglycan of articular cartilage . Like ADAMTS4, another member of the ADAMTS-family, ADAMTS5 cleaves aggrecan at 5 sites . Four cleavage sites are located in the chondroitin sulfate-rich region between aggrecan globular domains G2 and G3, while one site is contained in the rod-shaped polypeptide between globular domains G1 and G2. In addition, ADAMTS5 cleaves one more site in the chondroitin sulfate-rich region that is not cleaved by ADAMTS4 . ADAMTS5 is inhibited by TIMP 3 [5,6] and 2-macroglobulin .
Purity: As judged from SDS-PAGE truncated ADAMTS5 represents 50% of total protein of the preparation. The protein concentration is 0.1mg/ml.
Specific Activity: Aggrecanase activity of the ADAMTS5 preparation is determined with recombinant aggrecan interglobular domain (Aggrecan-IGD from MD Biosciences). ADAMTS4 hydrolyzes the “aggrecanase” site within this domain (peptide bond E373 -A374 in human aggrecan). The recombinant substrate is incubated at a concentration of 0.1 μM with ADAMTS5 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 μM leupeptin, 1 μM pepstatin, 1 mM Pefabloc, 0.05 % Brij 35 for 15 min at 37 °C. A series of ADAMTS5 dilutions ranging from 103- to 105 -fold is tested. 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, one directed against the neoepitope ARGSVIL, the other against the sequence C-terminal from the neoepitope (InviLISA Aggrecanase Activity Assay). Under the specified conditions the hydrolysis rate is > 0.5 nmoles hydrolyzed substrate/ min x ml ADAMTS5 preparation or > 5 nmoles hydrolyzed substrate/ min x mg.
Inhibitors: ADAMTS5 is inhibited by tissue inhibitor of matrix metalloproteinases 3 (TIMP3) and by 2-macroglobulin (see section 3). Enzyme activity is also suppressed by chelators of divalent cations as EDTA and by synthetic metalloproteinase inhibitors.
Stability & Storage: Recombinant ADAMTS5 is stable until the expiry date given on the label when stored at -70°C. The enzyme can be kept at -20°C for several weeks. Repeated freezing and thawing should be avoided.
Applications: Recombinant ADAMTS5 is used to study the degradation of extracellular matrix proteoglycans, to screen for inhibitors of ADAMTS5 and to characterise inhibitor actions. The enzyme preparation can also be utilized in enzymatic and immunochemical assays.