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-12) in Escherichia 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 buffer and purified by gel filtration chromatography under denaturing conditions. Partial refolding of the protein by dialysis into a 3 M urea buffer caused selective degradation of the truncated form of the protein, while the intact catalytic domain was unaffected by proteolysis. An SP-Sepharose chromatography step purified the protein to homogeneity and served also to complete the refolding. The purified protein was homogeneous by mass spectrometry and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a concentration 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-scale protein production for structural studies.

The effect of interleukin-6 and soluble interleukin-6 receptor protein on the bone resorptive activity of human osteoclasts generated in vitro.

The role of interleukin-6 (IL-6) in the regulation of bone resorption is controversial and has not been studied using human tissue in vitro. This study exploits a recently described in vitro model, whereby osteoclasts, defined as cells that resorb bone, can be generated from human bone marrow, and investigated the effect of IL-6 and its soluble receptor on bone resorption, in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2 vitamin D3]. Human bone marrow was cultured to form a confluent stroma, sedimented onto devitalized bone slices, and recharged with non-adherent bone marrow cells. 1,25(OH)2 vitamin D3 increased bone resorption, whereas IL-6 failed to induce a similar stimulatory effect. Both IL-6 at 100 ng/ml and soluble IL-6 receptor protein in the absence of exogenous IL-6 inhibited the stimulatory effect of 1,25(OH)2 vitamin D3. Bone resorption was never observed when non-adherent haemopoietic cells were cultured in the absence of stroma but in the presence of IL-6, which indicates that IL-6 cannot replace the stromal factor(s) required for the formation of cells capable of resorbing bone. These results suggest that IL-6 at high concentrations is not a critical cytokine in stimulating osteoclastic bone resorption.

The role of 1,25-dihydroxycholecalciferol and prostaglandin E2 in the regulation of human osteoclastic bone resorption in vitro.

Prostaglandins increase human osteoclast generation in vivo whereas they have been shown to exert the opposite effect in vitro: the latter results are based on enumeration of osteoclast-like cells, whose nature is controversial. We have generated human osteoclasts in vitro as assessed by bone resorption, a function unique to the osteoclast, and analysed the role of prostaglandin E2 (PGE2) in osteoclast activity. Human bone marrow cells were cultured to form a mature stroma and then sedimented onto bone slices with or without a recharge of non-adherent bone marrow cells. Bone resorption was increased by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and PGE2 and inhibited by indomethacin: this inhibition was reversed by addition of PGE2. Our work supports the observation that PGE2 increases bone resorption in vivo and demonstrates the value of assessing osteoclast generation and activity in vitro using bone resorption.