Synthesis and refolding of human TIMP-2 from E. coli, with specific activity for MMP-2.
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.
Negro
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Department of Biochemistry, School of Veterinary Medicine, University of Padova, Italy.
Onisto
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M
Masiero
L
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Garbisa
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S
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
D011506:Proteins; D011993:Recombinant Fusion Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2