title: Synthesis and refolding of human TIMP-2 from E. coli, with specific activity for MMP-2.
abstract: Tissue inhibitors of metalloproteinase (TIMPs) are inhibitory counterparts of collagenases, containing 12 cysteine residues paired to six internal disulphide bridges. TIMP-2, an inhibitory protein of 72 kDa gelatinase/type IV collagenase (MMP-2), was expressed in Escherichia coli as a fusion protein with a 34 amino acid NH2-linked tail containing six consecutive histidine residues. The protein was purified in a single-step using an ion metal affinity column (IMAC) in denaturing conditions. The immobilized fusion TIMP-2 protein was refolded at a high concentration in the column, producing about 5 mg of protein per litre of bacterial cells. It shows specific binding and inhibitory activity against MMP-2, but has no effect against 92 and 45 kDa gelatinases.
authors.affiliation: Department of Biochemistry, School of Veterinary Medicine, University of Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Negro
authors.forename: M
authors.initials: M
authors.lastname: Onisto
authors.forename: L
authors.initials: L
authors.lastname: Masiero
authors.forename: S
authors.initials: S
authors.lastname: Garbisa
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D018093:Gelatinases / Q000037:antagonists & inhibitors*; D006801:Humans; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2