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Synthesis and refolding of human TIMP‐2 from E. coli , with specific activity for MMP‐2
Author(s) -
Negro Alessandro,
Onisto Maurizio,
Masiero Laura,
Garbisa Spiridione
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00073-i
Subject(s) - gelatinases , collagenase , chemistry , gelatinase , escherichia coli , fusion protein , histidine , biochemistry , cysteine , affinity chromatography , metalloproteinase , inhibitory postsynaptic potential , matrix metalloproteinase , amino acid , microbiology and biotechnology , chromatography , enzyme , recombinant dna , biology , neuroscience , gene
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 NH 2 ‐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.