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Expression, purification and charaterization of recombinant mouse MT5‐MMP protein products
Author(s) -
Wang Xing,
Yi Jun,
Lei Jianxun,
Pei Duanqing
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01534-3
Subject(s) - matrix metalloproteinase , cell culture , transfection , recombinant dna , chemistry , metalloproteinase , microbiology and biotechnology , biochemistry , biophysics , biology , gene , genetics
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°C, but being relatively stable at temperatures 4°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.