Refolding and Recovery of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) from Inclusion Bodies Expressed by Escherichia coli.

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  • Oneda Hiroshi
    Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
  • Inouye Kuniyo
    Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

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Abstract

The recombinant prepro-form of human matrix metalloproteinase 7 (matrilysin or MMP-7) was overexpressed in Escherichia coli as insoluble inclusion bodies. The recombinant protein was refolded by 100-fold dilution after solubilization with 6M guanidine HCl. The refolding was monitored by the recovery of matrilysin activity. The addition of either 1.0M arginine or 0.1% Brij-35 promoted remarkably the refolding. The refolding was dependent on pH and temperature, with lower temperature (<10°C) and pH 6-8 preferable. Glutathione had no effect on refolding, and it was excluded from the refolding conditions. Starting with inclusion bodies (2.0g, wet) containing 360mg protein, 29.5mg of promatrilysin (30 kDa) was obtained after refolding with 1.0% Brij-35 at pH 7.5 and 4°C for 12 h. Pro-matrilysin (24.0mg) was purified to homogeneity by cation-exchange HPLC with a 15-fold increase in purity and an activity yield of 81.3%. Pro-matrilysin was converted entirely to matrilysin (19.0 kDa; 15.2mg) by activation with a mercuric reagent. The activity (kcat/Km) of matrilysin was 1.7×105M-1•s-1.

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