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.