title: Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli.
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 0.12mg/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.
authors: Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
authors: Andrezza N
authors: AN
authors: Gonçalves
authors: Cesar A
authors: CA
authors: Meschiari
authors: William G
authors: WG
authors: Stetler-Stevenson
authors: M Cristina
authors: MC
authors: Nonato
authors: Cleidson P
authors: CP
authors: Alves
authors: Enilza M
authors: EM
authors: Espreafico
authors: Raquel F
authors: RF
authors: Gerlach
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2