Kinetic Analysis of the Binding of Hemopexin-like Domain of Gelatinase B Cloned and Expressed in Pichia pastoris to Tissue Inhibitor of Metalloproteinases-1

Jörg Stute, Tayebeh Pourmotabbed, Harald Tschesche

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6 Citations (Scopus)

Abstract

The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.

Original languageEnglish (US)
Pages (from-to)509-514
Number of pages6
JournalJournal of Protein Chemistry
Volume22
Issue number6
DOIs
StatePublished - Aug 1 2003

Fingerprint

Hemopexin
Tissue Inhibitor of Metalloproteinase-1
Pichia
Matrix Metalloproteinase 9
Gelatinases
Tissue
Kinetics
Tissue Inhibitor of Metalloproteinases
Tissue Inhibitor of Metalloproteinase-2
Surface Plasmon Resonance
Matrix Metalloproteinase 2
Ion Exchange Chromatography
Surface plasmon resonance
Chromatography
Matrix Metalloproteinases
Gel Chromatography
Ion exchange
Gels
Metalloproteases

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Kinetic Analysis of the Binding of Hemopexin-like Domain of Gelatinase B Cloned and Expressed in Pichia pastoris to Tissue Inhibitor of Metalloproteinases-1",
abstract = "The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.",
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T1 - Kinetic Analysis of the Binding of Hemopexin-like Domain of Gelatinase B Cloned and Expressed in Pichia pastoris to Tissue Inhibitor of Metalloproteinases-1

AU - Stute, Jörg

AU - Pourmotabbed, Tayebeh

AU - Tschesche, Harald

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N2 - The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.

AB - The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.

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