Expression and purification of recombinant human indoleamine 2, 3-dioxygenase.
Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression.
Littlejohn
T K
TK
Australian Cataract Research Foundation, University of Wollongong, New South Wales, Australia.
Takikawa
O
O
Skylas
D
D
Jamie
J F
JF
Walker
M J
MJ
Truscott
R J
RJ
D006916:5-Hydroxytryptophan / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006639:Histidine; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D014366:Tryptophan Oxygenase