Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli

We have cloned, overexpressed, and purified the catalytic domain (residues Gly106 to Asn268) of human macrophage metalloelastase (MMP-l2) in Escherich ia coli. This construct represents a truncated form of the enzyme, lacking the N-terminal propeptide domain and the C-terminal hemopexin-like domain. The overexpressed protein was localized exclusively to insoluble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclusion bodies were solubilized in an 8 M guanidine-HCl b uffer and purified by gel filtration chromatography under denaturing condit ions. Partial refolding of the protein by dialysis into a 3 M urea buffer c aused selective degradation of the truncated form of the protein, while the intact catalytic domain was unaffected by proteolysis. An SP-Sepharose chr omatography seep purified the protein to homogeneity and served also to com plete the refolding. The purified protein was homogeneous by mass spectrome try and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a co ncentration of 9 mg/ml. This purification procedure enables the production of 23 mg of protein per liter of E. coli culture and is amenable to large-s cale protein production for structural studies. (C) 2000 Academic Press.