Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening.
Abstract
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.
Keywords:
(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.