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

Littlejohn, TK, Takikawa, O, Skylas, D, Jamie, JF, Walker, MJ and Truscott, RJW (2000) Expression and purification of recombinant human indoleamine 2,3-dioxygenase. Protein Expression and Purification, 19 1: 22-29. doi:10.1006/prep.2000.1214


Author Littlejohn, TK
Takikawa, O
Skylas, D
Jamie, JF
Walker, MJ
Truscott, RJW
Title Expression and purification of recombinant human indoleamine 2,3-dioxygenase
Journal name Protein Expression and Purification   Check publisher's open access policy
ISSN 1046-5928
Publication date 2000-06-01
Year available 2000
Sub-type Article (original research)
DOI 10.1006/prep.2000.1214
Open Access Status DOI
Volume 19
Issue 1
Start page 22
End page 29
Total pages 8
Place of publication SAN DIEGO
Publisher ACADEMIC PRESS INC ELSEVIER SCIENCE
Language eng
Abstract 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 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 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. (C) 2000 Academic Press.
Keyword Oxidative Tryptophan-Metabolism
Retrovirus-Infected Macaques
Single-Step Purification
Nitric-Oxide Synthase
Interferon-Gamma
Quinolinic Acid
Kynurenine Pathway
Ifn-Gamma
Indoleamine-2,3-Dioxygenase Activity
Competitive Inhibitors
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: ResearcherID Downloads - Archived
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 51 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 56 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Tue, 11 Oct 2011, 01:08:54 EST by System User