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Purification and Activity Determination of ADAMTS-4 and ADAMTS-5 and Their Domain Deleted Mutants.

Fowkes MM., Lim NH.

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.

More information Original publication

DOI

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

Type

Chapter

Publication Date

2020-01-01T00:00:00+00:00

Volume

2043

Pages

75 - 91

Total pages

16

Addresses

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Keywords

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