Expression and purification of recombinant human indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tr yptophan metabolism, has been implicated in the pathogenesis of many diseas es. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a f usion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosp hocellulose and nickel-agarose affinity chromatography, The yield of the fu sion protein was 1.4 mg per liter of bacterial culture with an overall reco very of 56% from the crude extract, When the culture medium was supplemente d with 7 mu M hemin, the purified protein contained 0.8 mol of heme per mob of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compar ed with 6.9 for the native enzyme. This was as expected from the addition o f the hexahistidyl tag. Similar to the native enzyme, the recombinant enzym e required methylene blue and ascorbic acid for enzyme activity and oxidize d not only L-tryptophan but also D-tryptophan and 5-hydroxy-L-tryptophan. T he molecular activities for these substrates and their K-m values were simi lar to those of the native enzyme, indicating that the addition of the hexa histidyl tag did not significantly affect catalytic activity. The recombina nt protein can therefore be used to investigate properties of the native en zyme. This will aid the development of specific inhibitors of indoleamine 2 ,3-dioxygenase, which may be effective in halting disease progression. (C) 2000 Academic Press.