A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.
Chapter
2020-01-01T00:00:00+00:00
2043
75 - 91
16
Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.
Cartilage, Extracellular Matrix, Humans, Culture Media, Chromatography, Affinity, Fluorescence Resonance Energy Transfer, Cell Culture Techniques, Catalytic Domain, Mutation, HEK293 Cells, ADAMTS4 Protein, ADAMTS5 Protein, Protein Domains