Kinetic analysis of the binding of hemopexin-like domain of gelatinase B cloned and expressed in Pichia pastoris to tissue inhibitor of metalloproteinases-1.
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.
Stute
Jörg
J
Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Bielefeld, Germany.
Pourmotabbed
Tayebeh
T
Tschesche
Harald
H
D006466:Hemopexin / Q000737:chemistry*; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*
D019715:Tissue Inhibitor of Metalloproteinase-1; D006466:Hemopexin; D020780:Matrix Metalloproteinase 9