Production of recombinant human matrix metalloproteinase 7 (Matrilysin) with potential role in tumor invasion by refolding from Escherichia coli inclusion bodies and development of sandwich ELISA of MMP-7

Metastatic potential of prostate cancer is thought to correlate with the degradation of basement membrane components by matrix metalloproteinases (MMPs). The MMP-7 (matrilysin) gene is overexpressed in prostate cancer as well as colorectum and brain cancer. In order to clarify the relation of MMP-7 to clinical stages of prostate cancer, recombinant human MMP-7 was produced to prepare antibodies for immunohistochemistry and immunoassay. Preproform of human MMP-7 was produced in Escherichia coli as inclusion bodies that could be solubilized and refolded to yield an activatable proenzyme. PreproMMP-7 (Mr 31,000) solubilized from inclusion bodies was converted to proMMP-7 (Mr 30,000) during the refolding steps. The refolded proMMP-7 was purified to about 80% homogeneity as MMP-7 by sequential ion-exchange and molecular-sieve chromatography. The active, mature form of MMP-7 (Mr 20,000) could be produced from proforms of MMP-7 by treatment with p-aminophenylmercuric acetate. Activated MMP-7 was shown to have proteolytic activity to fibronectin, casein, and diazotized, denatured collagen (Azocoll). Specific activity, as assayed with the denatured collagen as substrate, was measured to be about 3,100 units/mg protein of mature enzyme. Using recombinant proMMP-7 as antigen, monoclonal and polyclonal antibodies were prepared. A sandwich ELISA was developed using monoclonal antibody as the capture antibody and rabbit anti-proMMP-7 polyclonal IgG conjugated with biotin as the detection antibody; MMP-7 at 10 ng/ml was significantly detectable. The assay system is applicable on the measurement of MMP-7 levels in the clinical and pathologic specimens including serum from patients with different stages in malignancy of prostate cancer. These antibodies are useful for the retrospective analyses of prostate cancer on the basis of immunohistochemical evaluation.