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Purification of matrix metalloproteinases by column chromatography

Nature Protocols volume 3, pages 1111–1124 (2008)Cite this article

Abstract

Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin–Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG–Sepharose columns) and the final step for further purification (IgG–Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes ∼7–10 d.

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Figure 1: The domain structures of secreted-type matrix metalloproteinases (MMPs) and membrane-anchored MMPs.
Figure 2: Purification steps of proMMPs and matrix metalloproteinase (MMP).
Figure 3: Green A Dyematrex gel chromatograph for the purification of proMMP-3.
Figure 4: SDS-PAGE analysis of the samples from each step for proMMP-9 purification.
Figure 5: Schematic illustration of a linear gradient mixer.

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Acknowledgements

We are grateful to all the previous and current members of Y.O.'s lab who contributed to the development of these techniques. Y.O. was supported by the grant-in-aid from the Ministry of Education, Science and Culture of Japan (19109004).

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  1. Department of Biochemistry, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo, 102-8159, Japan

    Kazushi Imai

  2. Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-0016, Japan

    Yasunori Okada

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Correspondence to Yasunori Okada.

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Imai, K., Okada, Y. Purification of matrix metalloproteinases by column chromatography. Nat Protoc 3, 1111–1124 (2008). https://doi.org/10.1038/nprot.2008.74

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