title: Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli.
abstract: Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 μM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme's activity without any aggregation.
authors.affiliation: Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Sara
authors.initials: S
authors.lastname: Mohseni
authors.affiliation: Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Tahereh Tohidi
authors.initials: TT
authors.lastname: Moghadam
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Bahareh
authors.initials: B
authors.lastname: Dabirmanesh
authors.affiliation: Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran; Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address: khajeh@modares.ac.ir.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014508:Urea / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D014508:Urea; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: Active full length recombinant human gelatinase MMP-9 (rh MMP-9); Inclusion body; Matrix metalloproteinases (MMPs); Refolding