Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli |
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Authors: | Gonçalves Andrezza N Meschiari Cesar A Stetler-Stevenson William G Nonato M Cristina Alves Cleidson P Espreafico Enilza M Gerlach Raquel F |
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Affiliation: | a Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil b Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil c Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA d Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil e Department of Molecular and Cellular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil f Department of Morphology, Stomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil |
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Abstract: | Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 °C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. |
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Keywords: | MMPs, matrix metalloproteinases TIMP, tissue inhibitor of metalloproteinase FN-II, fibronectin type II |
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