Expression, purification and characterization of recombinant mouse MT5-MMP protein products.
We have recently identified the fifth member of the membrane-type matrix metalloproteinase subfamily, MT5-MMP/MMP24, which is expressed in a brain specific manner (Duanqing Pei (1999) J. Biol. Chem. 274, 8925-8932). To further characterize its enzymic properties, an expression construct was engineered to produce MT5-MMP as a soluble and active form by truncating its transmembrane domain. Stable expression cell lines were subsequently established from MDCK cells transfected with this construct. Unfortunately, purification of MT5-MMP from the culture media in large quantity proves to be difficult initially due to its rapid turnover via a mechanism which can be inhibited by a broad spectrum metalloproteinase inhibitor, BB94. Thus, BB94 was included in the cell culture medium and throughout the purification process except the final step of chromatography to protect MT5-MMP from destruction. Purified to homogeneity and free of the synthetic inhibitor, MT5-MMP can activate progelatinase A efficiently in a TIMP2 sensitive fashion. A preliminary screen for its potential substrates among extracellular matrix components identified the proteoglycans as the preferred substrates for MT5-MMP. Furthermore, it is determined that the stability of purified MT5-MMP is temperature dependent with rapid destruction at 37 degrees C, but being relatively stable at temperatures 4 degrees C or lower. These observations establish MT5-MMP as a proteoglycanase with a short half-life at body temperature, which may be critical for tightly controlled turnover of ECM components such as those in the brain.
Wang
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X
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
Yi
J
J
Lei
J
J
Pei
D
D
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004285:Dogs; D004795:Enzyme Stability; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C118502:Mmp24 protein, mouse