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Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening.

AIM
To obtain a catalytically active human macrophage elastase catalytic domain (hMECD) and to establish an efficient high-throughput method for screening macrophage elastase inhibitors.


METHODS
Catalytic domain of human macrophage elastase was expressed in E coli and characterized to establish a high-throughput screening assay using a colorimetric method. A set of 8560 pure compounds and mixtures were screened.


RESULTS
We have constructed an efficient E coli system for this human protein expression, and the recombinant hMECD protein was purified to homogeneity using anion-exchange chromatography after in vitro refolding from inclusion bodies. The yield of active hMECD protein was 23 mg from one liter of E coli culture after purification. Calcium and zinc ions were required both in refolding and enzymatic activity, but high concentration of zinc inhibited the refolding and activity. The hMECD cleaved several synthetic substrates including a chromogenic thiopeptolide and fluorogenic peptides with optimal activity around pH 8.0. Screening of 8560 compounds and mixtures led to identify 27 pure compounds and 14 natural products with inhibitory activity higher than 80 % at 20 mg/L.


CONCLUSION
An efficient expression and purification method for hMECD protein has been established, and the assay is effective, reliable, and fast in identifying the recombinant protein inhibitors.