Expression, purification and charaterization of recombinant mouse MT5-MMP protein products

We have recently identified the fifth member of the membrane-type matrix me talloproteinase subfamily, MT5-MMP/MMP24, which is expressed in a brain spe cific manner (Duanqing Pei (1999) J, Biol, Chem, 274, 8925-8932). To furthe r characterize its enzymic properties, an expression construct was engineer ed to produce MT5-MMP as a soluble and active form by truncating its transm embrane domain, Stable expression cell lines were subsequently established from MDCK cells transfected with this construct. Unfortunately, purificatio n of MT5-MMP from the culture media in large quantity proves to be difficul t initially due to its rapid turnover via a mechanism which can be inhibite d by a broad spectrum metalloproteinase inhibitor, BB94, Thus, BB94 was inc luded in the cell culture medium and throughout the purification process ex cept the final step of chromatography to protect MT5-MMP from destruction. Purified to homogeneity and free of the synthetic inhibitor, MT5-MMP can ac tivate progelatinase A efficiently in a TIMP2 sensitive fashion. A prelimin ary screen for its potential substrates among extracellular matrix componen ts identified the proteoglycans as the preferred substrates for MT5-MMP, Fu rthermore, it is determined that the stability of purified MT5-MMP is tempe rature dependent with rapid destruction at 37 degrees C, but being relative ly stable at temperatures 4 degrees C or lower. These observations establis h MT5-MMP as a proteoglycanase with a short half-life at body temperature, which may be critical for tightly controlled turnover of ECM components suc h as those in the brain. (C) 1999 Federation of European Biochemical Societ ies.