Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase.
The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 degrees C, with reduced formation at 30 degrees C. The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees C and purification by nickel-agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 micromol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3-5 mg/L of bacterial culture) and heme content are greater than previously reported.
Austin
Christopher J D
CJ
Department of Chemistry, Macquarie University, Sydney, Australia.
Mizdrak
Jasminka
J
Matin
Azadeh
A
Sirijovski
Nicholche
N
Kosim-Satyaputra
Priambudi
P
Willows
Robert D
RD
Roberts
Thomas H
TH
Truscott
Roger J W
RJ
Polekhina
Galina
G
Parker
Michael W
MW
Jamie
Joanne F
JF
D000622:Aminolevulinic Acid / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007501:Iron / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D007737:Kynurenine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011524:Protoporphyrins / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014366:Tryptophan Oxygenase / Q000096:biosynthesis* / Q000737:chemistry*
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D011524:Protoporphyrins; D011994:Recombinant Proteins; D007737:Kynurenine; D007544:Isopropyl Thiogalactoside; D006418:Heme; D006639:Histidine; D000622:Aminolevulinic Acid; D014364:Tryptophan; C028025:protoporphyrin IX; D007501:Iron; D014366:Tryptophan Oxygenase