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KMID : 0578320020130010118
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Molecules and Cells
2002 Volume.13 No. 1 p.118 ~ p.124
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Refolding of the Catalytic and Hinge Domains of Human MT1-MMP Expressed in Escherichia coli and Its Characterization
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Koo Hyun-Min
Kim Joo-Hyon
Hwang In-Kwan
Lee Seo-Jin
Kim Tae-Han
Rhee Ki-Hyeong
Lee Seung-Taek
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Abstract
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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 al-most exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chroma-tography 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 spec-trometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methion-ine and Ile114-Thr299, and that the mature form cor-responds 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.
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KEYWORD
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Autoproteolysis, Catalytic Domain, Membrane Type-1 Matrix Metalloproteinase (MT1-MMP), Refolding
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