Construction, Expression, and Characterization of a Baculovirally Expressed Catalytic Domain of Human Matrix Metalloproteinase-9 |
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Authors: | Sepideh Sadatmansoori John MacDougall Shahram Khademi Laurence S Cooke Linda Guarino Edgar F Meyer Reza Forough |
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Institution: | Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA. |
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Abstract: | We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate. |
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Keywords: | MMP-9 expression baculovirus minienzyme characterization |
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