This paper describes the first report of the production and use of an artificial recombinant protein substrate to study "aggrecanase" activity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the "marker" sequences FLAG TM at the amino terminus and the human immunoglobulin G1 constant region at the carboxyl terminus. The expressed protein occurs as large multimolecular aggregates (>120 kDa) that, upon reduction, consist of a major isoform of 72 kDa (containing the IGD) and a minor 39-kDa species that through alternative splicing has had the IGD deleted. Using this recombinant substrate we developed a novel agarose cell culture system containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrecanase activities. These studies showed the following.

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