Refolding of the catalytic and hinge domains of human MT1-MMP expressed in Escherichia coli and its characterization

Hyun Min Koo, Joo Hyon Kim, In Kwan Hwang, Seo Jin Lee, Tae Han Kim, Ki Hyeong Rhee, Seung Taek Lee

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12 Citations (Scopus)


The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli. The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea. During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form. By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290. These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding. Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay. In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2. These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP.

Original languageEnglish
Pages (from-to)118-124
Number of pages7
JournalMolecules and cells
Issue number1
Publication statusPublished - 2002 Feb

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology


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