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

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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
Volume13
Issue number1
Publication statusPublished - 2002 Feb

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Refolding of the catalytic and hinge domains of human MT1-MMP expressed in Escherichia coli and its characterization'. Together they form a unique fingerprint.

  • Cite this

    Koo, H. M., Kim, J. H., Hwang, I. K., Lee, S. J., Kim, T. H., Rhee, K. H., & Lee, S. T. (2002). Refolding of the catalytic and hinge domains of human MT1-MMP expressed in Escherichia coli and its characterization. Molecules and cells, 13(1), 118-124.