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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.

Original publication

DOI

10.1007/978-1-4939-9698-8_7

Type

Chapter

Publication Date

2020

Volume

2043

Pages

75 - 91

Keywords

ADAMTS, Active-site titrations, Affinity chromatography, Aggrecanase probes, FRET substrates, Proteinase purification, ADAMTS4 Protein, ADAMTS5 Protein, Cartilage, Catalytic Domain, Cell Culture Techniques, Chromatography, Affinity, Culture Media, Extracellular Matrix, Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Mutation, Protein Domains